U.S. patent application number 11/401093 was filed with the patent office on 2006-10-12 for methods for treating infectious disease exacerbated asthma.
This patent application is currently assigned to Coley Pharmaceutical Group, Inc.. Invention is credited to George Tilo De Sanctis, Raymond Anthony Jupp, Arthur M. Krieg, John A. JR. Schmidt, Stephen Leslie Underwood.
Application Number | 20060229271 11/401093 |
Document ID | / |
Family ID | 36934412 |
Filed Date | 2006-10-12 |
United States Patent
Application |
20060229271 |
Kind Code |
A1 |
Krieg; Arthur M. ; et
al. |
October 12, 2006 |
Methods for treating infectious disease exacerbated asthma
Abstract
The invention relates to methods of treating infectious disease
exacerbated asthma. In particular, the methods are useful for
treating viral exacerbated asthma using CpG oligonucleotides.
Inventors: |
Krieg; Arthur M.;
(Wellesley, MA) ; De Sanctis; George Tilo;
(Skillman, NJ) ; Underwood; Stephen Leslie;
(Bedminster, NJ) ; Jupp; Raymond Anthony;
(Chatham, NJ) ; Schmidt; John A. JR.; (Green
Brook, NJ) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, PC
FEDERAL RESERVE PLAZA
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Assignee: |
Coley Pharmaceutical Group,
Inc.
Wellesley
MA
Sanofi-Aventis U.S.P. LLC
Bridgewater
NJ
|
Family ID: |
36934412 |
Appl. No.: |
11/401093 |
Filed: |
April 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60669548 |
Apr 8, 2005 |
|
|
|
Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61P 31/16 20180101;
A61K 31/7088 20130101; C12N 15/117 20130101; A61K 39/39 20130101;
A61P 37/04 20180101; A61K 2039/55561 20130101; A61K 31/713
20130101; C12N 2310/17 20130101; A61P 11/06 20180101 |
Class at
Publication: |
514/044 |
International
Class: |
A61K 48/00 20060101
A61K048/00 |
Claims
1. A method for treating viral exacerbated asthma, comprising:
administering to an asthmatic subject an effective amount of a
C-class CpG oligonucleotide for treating viral exacerbated
asthma.
2. The method of claim 1, wherein the viral exacerbated asthma is
caused by a respiratory virus.
3. The method of claim 2, wherein the respiratory virus is not
RSV.
4. The method of claim 1, wherein the viral exacerbated asthma is
caused by influenza virus.
5. The method of claim 1, wherein the subject is identified by a
medical worker.
6. The method of claim 1, wherein the subject is identified based
on exposure to a risk factor for viral infection.
7. The method of claim 1, wherein the method includes the step of
identifying an asthmatic subject at risk of viral infection.
8. The method of claim 1, wherein the C-class oligonucleotide is a
semi-soft oligonucleotide.
9. The method of claim 1, wherein the C-class oligonucleotide is
SEQ ID NO: 10.
10. A method for treating viral exacerbated asthma, comprising:
identifying an asthmatic subject at risk of viral infection, and
administering to the asthmatic subject an effective amount of a CpG
oligonucleotide for treating viral exacerbated asthma.
11. The method of claim 10, wherein the viral exacerbated asthma is
caused by a respiratory virus.
12. The method of claim 11, wherein the respiratory virus is not
RSV.
13. The method of claim 10, wherein the viral exacerbated asthma is
caused by influenza virus.
14. The method of claim 10, wherein the risk factor is influenza
season.
15. The method of claim 10, wherein the risk factor is travel to a
destination with a high risk of viral exposure.
16. The method of claim 10, wherein the CpG oligonucleotide is a
C-class oligonucleotide.
17. The method of claim 16, wherein the C-class oligonucleotide is
a semi-soft oligonucleotide.
18. The method of claim 16, wherein the C-class oligonucleotide is
SEQ ID NO:10.
19. A method for treating viral exacerbated asthma, comprising:
administering to an asthmatic subject undergoing a non-CpG asthma
therapy an effective amount of a CpG oligonucleotide for treating
viral exacerbated asthma.
20. The method of claim 19, wherein the non-CpG asthma therapy is
steroid therapy.
21. The method of claim 19, wherein the non-CpG asthma therapy is
administered at a different time than the CpG oligonucleotide.
22. The method of claim 19, wherein the non-CpG asthma therapy is
administered at the same time as the CpG oligonucleotide.
23. The method of claim 19, wherein the CpG oligonucleotide is a
C-class oligonucleotide.
24. The method of claim 23, wherein the C-class oligonucleotide is
a semi-soft oligonucleotide.
25. The method of claim 23, wherein the C-class oligonucleotide is
SEQ ID NO:10.
26. A method for treating infectious disease exacerbated asthma,
comprising: identifying an asthmatic subject at risk of infection,
and administering to the asthmatic subject an effective amount of a
CpG oligonucleotide for treating infectious disease exacerbated
asthma.
27. A method for treating viral exacerbated asthma, comprising:
identifying an asthmatic subject at risk of viral infection, and
administering to the asthmatic subject a CpG oligonucleotide in an
amount that is sub-therapeutic for treating viral infection,
wherein the CpG oligonucleotide is effective for reducing immune
cell accumulation.
28. The method of claim 27, wherein the immune cell is a
neutrophil.
29. The method of claim 27, wherein the immune cell is an
eosinophil
30. A method for treating viral exacerbated asthma, comprising:
identifying an asthmatic subject at risk of viral infection, and
administering to the asthmatic subject at least three doses of CpG
oligonucleotide, wherein the at least three doses of CpG
oligonucleotide are temporally separated from one another by at
least three days.
31. A method for treating viral exacerbated asthma, comprising:
identifying a risk factor for viral infection, and administering to
an asthmatic subject an effective amount of a CpG oligonucleotide
for treating viral exacerbated asthma during a period of time when
the asthmatic subject is at risk of viral infection.
Description
RELATED APPLICATION
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. Provisional Application No. 60/669,584, filed Apr.
8, 2005, entitled METHODS FOR TREATING INFECTIOUS DISEASE
EXACERBATED ASTHMA, the entire contents of which is hereby
incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to methods of
treating asthma that is exacerbated by infectious disease using
immunostimulatory oligonucleotides, as well as compositions
thereof.
BACKGROUND OF THE INVENTION
[0003] Bacterial DNA has immune stimulatory effects to activate B
cells and natural killer cells, but vertebrate DNA does not
(Tokunaga, T., et al., 1988. Jpn. J. Cancer Res. 79:682-686;
Tokunaga, T., et al., 1984, JNCI 72:955-962; Messina, J. P., et
al., 1991, J. Immunol. 147:1759-1764; and reviewed in Krieg, 1998,
In: Applied Oligonucleotide Technology, C. A. Stein and A. M.
Krieg, (Eds.), John Wiley and Sons, Inc., New York, N.Y., pp.
431-448). It is now understood that these immune stimulatory
effects of bacterial DNA are a result of the presence of
unmethylated CpG dinucleotides in particular base contexts (CpG
motifs), which are common in bacterial DNA, but methylated and
underrepresented in vertebrate DNA (Krieg et al, 1995 Nature
374:546-549; Krieg, 1999 Biochim. Biophys. Acta 93321:1-10). The
immune stimulatory effects of bacterial DNA can be mimicked with
synthetic oligodeoxynucleotides (ODN) containing these CpG motifs.
Such CpG ODN have highly stimulatory effects on human and murine
leukocytes, inducing B cell proliferation; cytokine and
immunoglobulin secretion; natural killer (NK) cell lytic activity
and IFN-.gamma. secretion; and activation of dendritic cells (DCs)
and other antigen presenting cells to express costimulatory
molecules and secrete cytokines, especially the Th1-like cytokines
that are important in promoting the development of Th1-like T cell
responses. These immune stimulatory effects of native
phosphodiester backbone CpG ODN are highly CpG specific in that the
effects are dramatically reduced if the CpG motif is methylated,
changed to a GpC, or otherwise eliminated or altered (Krieg et al,
1995 Nature 374:546-549; Hartmann et al, 1999 Proc. Natl. Acad. Sci
USA 96:9305-10).
[0004] In early studies, it was thought that the immune stimulatory
CpG motif followed the formula
purine-purine-CpG-pyrimidine-pyrimidine (Krieg et al, 1995 Nature
374:546-549; Pisetsky, 1996 J. Immunol. 156:421-423; Hacker et al.,
1998 EMBO J. 17:6230-6240; Lipford et al, 1998 Trends in Microbiol.
6:496-500). However, it is now clear that mouse lymphocytes respond
quite well to phosphodiester CpG motifs that do not follow this
"formula" (Yi et al., 1998 J. Immunol. 160:5898-5906) and the same
is true of human B cells and dendritic cells (Hartmann et al, 1999
Proc. Natl. Acad. Sci USA 96:9305-10; Liang, 1996 J. Clin. Invest.
98:1119-1129).
[0005] Several different classes of CpG oligonucleotides has
recently been described. One class is potent for activating B cells
but is relatively weak in inducing IFN-.alpha. and NK cell
activation; this class has been termed the B class. The B class CpG
oligonucleotides typically are fully stabilized and include an
unmethylated CpG dinucleotide within certain preferred base
contexts. See, e.g., U.S. Pat. Nos. 6,194,388; 6,207,646;
6,214,806; 6,218,371; 6,239,116; and 6,339,068. Another class of
CpG oligonucleotides activates B cells and NK cells and induces
IFN-.alpha.; this class has been termed the C-class. The C-class
CpG oligonucleotides, as first characterized, typically are fully
stabilized, include a B class-type sequence and a GC-rich
palindrome or near-palindrome. This class has been described in
co-pending U.S. provisional patent application 60/313,273, filed
Aug. 17, 2001 and US10/224,523 filed on Aug. 19, 2002 and related
PCT Patent Application PCT/US02/26468 published under International
Publication Number WO 03/015711.
SUMMARY OF THE INVENTION
[0006] It has been discovered herein that CpG oligonucleotides (CpG
ODN) are particularly effective in combating infections, and
particularly upper respiratory tract virus, that are a cause of
asthma exacerbations. In some aspects of the invention C-class CpG
ODN are particularly effective for carrying out the methods. As
shown in the Examples below, C-class CpG ODN induced a panel of
IFN-associated genes in the mouse, including those for anti-viral
proteins, and protected against airway inflammation exacerbated by
combined antigen and virus exposures.
[0007] In some aspects the invention relates to a method for
treating viral exacerbated asthma, by administering to an asthmatic
subject an effective amount of a C-class CpG oligonucleotide for
treating viral exacerbated asthma.
[0008] In other aspects the invention relates to a method for
treating viral exacerbated asthma by identifying an asthmatic
subject at risk of viral infection, and administering to the
asthmatic subject an effective amount of a CpG oligonucleotide for
treating viral exacerbated asthma. The subject may be identified by
a medical worker. In other embodiments the subject is identified
based on exposure to a risk factor for viral infection.
[0009] According to other aspects the invention is a method for
treating viral exacerbated asthma by administering to an asthmatic
subject undergoing a non-CpG asthma therapy an effective amount of
a CpG oligonucleotide for treating viral exacerbated asthma. The
non-CpG asthma therapy may be a steroid therapy. In some
embodiments the non-CpG asthma therapy is administered at a
different time than the CpG oligonucleotide. In other embodiments
the non-CpG asthma therapy is administered at the same time as the
CpG oligonucleotide.
[0010] A method for treating infectious disease exacerbated asthma
by identifying an asthmatic subject at risk of infection, and
administering to the asthmatic subject an effective amount of a CpG
oligonucleotide for treating infectious disease exacerbated asthma
is provided according to other aspects of the invention.
[0011] In another aspect the invention is a method for treating
viral exacerbated asthma, by identifying a risk factor for viral
infection, and administering to an asthmatic subject an effective
amount of a CpG oligonucleotide for treating viral exacerbated
asthma during a period of time when the asthmatic subject is at
risk of viral infection. In some embodiments the risk factor is
influenza season. In other embodiments the risk factor is travel to
a destination with a high risk of viral exposure.
[0012] In some embodiments the viral exacerbated asthma is caused
by a respiratory virus. Optionally the respiratory virus is not
RSV. In other embodiments the viral exacerbated asthma is caused by
influenza virus.
[0013] The CpG oligonucleotide in some embodiments is a C-class
oligonucleotide. The C-class oligonucleotide may optionally be a
semi-soft oligonucleotide, such as, for instance, SEQ ID NO:
10.
[0014] A method for treating viral exacerbated asthma, by
identifying an asthmatic subject at risk of viral infection, and
administering to the asthmatic subject a CpG oligonucleotide in an
amount that is sub-therapeutic for treating viral infection,
wherein the CpG oligonucleotide is effective for reducing immune
cell accumulation is also provided. The immune cell may be, for
instance, a neutrophil or an eosinophil.
[0015] In other aspects the invention is a method for treating
viral exacerbated asthma, by identifying an asthmatic subject at
risk of viral infection, and administering to the asthmatic subject
at least three doses of CpG oligonucleotide, wherein the at least
three doses of CpG oligonucleotide are temporally separated from
one another by at least three days. In some embodiments the doses
are separated from one another by 1 week, 2 weeks, 3 weeks, one
month, one year or any integer value there between.
[0016] Use of an oligonucleotide of the invention for stimulating
an immune response and or the treatment of viral exacerbated asthma
is also provided as an aspect of the invention.
[0017] A method for manufacturing a medicament of an
oligonucleotide of the invention for stimulating an immune response
and or the treatment of viral exacerbated asthma is also
provided.
[0018] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention. This invention is not limited in its application
to the details of construction and the arrangement of components
set forth in the following description or illustrated in the
drawings. The invention is capable of other embodiments and of
being practiced or of being carried out in various ways. Also, the
phraseology and terminology used herein is for the purpose of
description and should not be regarded as limiting. The use of
"including," "comprising," or "having," "containing", "involving",
and variations thereof herein, is meant to encompass the items
listed thereafter and equivalents thereof as well as additional
items.
BRIEF DESCRIPTION OF DRAWINGS
[0019] The figures are illustrative only and are not required for
enablement of the invention disclosed herein.
[0020] FIG. 1 is a schematic of an abbreviated study schedule
showing some of the experimental conditions carried out in Example
1 and 2.
[0021] FIG. 2 is a schematic of a detailed study schedule showing
an experimental condition carried out in Example 1 (#3).
[0022] FIG. 3 is a series of graphs depicting IFN-.alpha. ((FIG.
3a), IFN-.gamma. (FIG. 3b), and IP-10 (FIG. 3c) induction, and a
second series of graphs depicting the upregulation of for
2'5'-oligoadenylate synthetase (FIG. 3d), Mx1 (FIG. 3e), and
indoleamine 2,3-dioxygenase (FIG. 3f) in mouse lung. The x-axes
represent .mu.g of oligonucleotide per kg of mouse. The y-axes
represent cytokine in pg/ml (FIGS. 3a-3c) or the amount of RNA as a
ratio of GAPDH RNA (FIGS. 3d-3f).
[0023] FIG. 4a is a graph depicting viral nuclear protein titer in
mouse lungs. The x-axis represents .mu.g of oligonucleotide per kg
of mouse (infected or uninfected) and the y-axis represents
absorbance. FIGS. 4b and 4c are graphs showing neutrophils and
mononuclear cells, respectively, that are present in
bronchoalveolar lavage fluid. The x-axes represent .mu.g of
oligonucleotide per kg of mouse (infected or uninfected) and the
y-axes represent numbers of cells.times.10.sup.3/ml.
[0024] FIG. 5 is a series of graphs depicting total cells
accumulated in response to treatment, including total leukocytes
(FIG. 5a), neutrophils (FIG. 5b), and mononuclear cells (FIG. 5c)
in bronchoaveolar lavage fluid in antigen challenged and virus
infected mice. The x-axes represent challenge categories of mice
and the y-axes represent numbers of cells.times.10.sup.6/ml (5a) or
.times.10.sup.3/ml (5b and 5c).
[0025] FIG. 6a is a graph depicting methacholine-induced increase
in airway resistance. The x-axis represents mg/ml methacholine and
the y-axis represents airway resistance as % of unchallenged
control. FIG. 6b shows the baseline airway resistance. FIG. 6c
shows areas under the methacholine dose-response curve. Results are
presented as mean.+-.SEM (n=7-9).). * P<0.05 compared with group
indicated (Mann-Whitney two-tailed test).
[0026] FIG. 7 is a series of graphs depicting total cells
accumulated in response to treatment, including total leukocytes
(FIG. 7a), eosinophils (FIG. 7b), neutrophils (FIG. 7c), and
mononuclear cells (FIG. 7d) as well as mouse body weight (FIG. 7e).
The x-axes represent challenge categories of mice.
[0027] FIG. 8 is a series of graphs demonstrating induction of
TLR9-associated cytokines in mouse airways in vivo. FIG. 8a shows
IFN.alpha., FIG. 8b shows IFN.gamma., FIG. 8c shows IP-10, FIG. 8d
shows IL-6, and FIG. 8e shows IL-12p40. Results are presented as
mean.+-.SEM (n=10). The x-axes represent .mu.g of oligonucleotide
per kg of mouse and the y-axes represent cytokine concentration in
pg/ml.
[0028] FIG. 9 is a series of graphs demonstrating induction of
cytokines ex vivo.
[0029] FIG. 9a shows IL-5, FIG. 9b shows IL-13, and FIG. 9c shows
IFN.gamma.. Results are presented as mean.+-.SEM (n=7-8). *
P<0.05 compared with vehicle-treated group (Kruskal-Wallis test
followed by Dunn's test for multiple comparisons). The x-axes
represent .mu.g of oligonucleotide per kg of mouse and the y-axes
represent cytokine concentration in pg/ml.
[0030] FIG. 10 is two graphs showing suppression of antigen-induced
accumulations of eosinophils and lymphocytes in mouse airways in
vivo by SEQ ID NO: 10. FIG. 10a shows IgE production and FIG. 10b
shows IgG2a production. Results are presented as mean.+-.SEM
(n=9-10). * P<0.05 compared with vehicle-treated group
(Kruskal-Wallis test followed by Dunn's test for multiple
comparisons). The x-axes represent .mu.g of oligonucleotide per kg
of mouse (sensitized or unsensitized) and the y-axes represent
absorbance units as a measurement of serum antibody titer.
[0031] FIG. 11 is four graphs demonstrating the accumulations of
eosinophils and lymphocytes in mouse airways in vivo after
administration of SEQ ID NO: 10. FIG. 11a shows total leukocytes
present, FIG. 11b shows eosinophils, FIG. 11c shows CD4-positive T
cells, and FIG. 11d shows B cells. Results are presented as
mean.+-.SEM (n=6). * P<0.05 compared with vehicle-treated group
(Kruskal-Wallis test followed by Dunn's test for multiple
comparisons). The x-axes represent .mu.g of oligonucleotide per kg
of mouse (sensitized or unsensitized) and the y-axes represent
number of cells.
DETAILED DESCRIPTION
[0032] Toll-like receptor 9 (TLR9) allows discrete populations of
immune cells to recognize unmethylated CpG oligodeoxynucleotides or
oligonucleotides (CpG ODN) and to activate host defense mechanisms
and initiate immune effects, resulting in suppressed Th2-type
responses. Different classes of CpG ODN have been described on the
basis of structure and activity characteristics. C-class CpG ODN
generally have a 5' end stimulatory sequence containing at least
one CpG motif, and a GC-rich palindrome. C-class CpG ODN induce
very high titers of interferon alpha (IFN.alpha.) from immune
cells.
[0033] According to some aspects of the invention, it has been
discovered that C-class CpG ODN are of particular value as a novel
therapy for upper respiratory tract infections and preferably viral
infections as they exacerbate allergic asthma. The data presented
in the Examples below, have demonstrated that when dosed into mouse
airways, a C-class CpG ODN can induce IFN-associated genes known to
have immune-modifying and/or anti-viral activities. In particular,
2'5'-oligoadenylate synthetase and Mx1 (mouse homologue of MxA) are
known to have marked anti-viral activity. In our mouse models, a
C-class CpG ODN showed protective effects against influenza
infection, and suppressed the exacerbated airway inflammation
induced by combined antigen challenge and influenza infection.
[0034] Thus, in some aspects the invention relates to methods for
treating infectious disease exacerbated asthma, and in particular
viral exacerbated asthma. Bacterial, viral, and fungal infections
exacerbate and/or induce asthma. Infectious disease exacerbated
asthma is a condition which occurs in an asthmatic subject. The
asthmatic subject, one who has been diagnosed with asthma or is
otherwise susceptible to asthma, when exposed to an infectious
agent experiences an asthmatic response or an existing/ongoing
asthmatic attack is worsened.
[0035] Thus, the invention in one aspect involves the finding that
CpG immunostimulatory oligonucleotides are useful in treating
infectious disease exacerbated asthma.
[0036] In some embodiments the subject is at risk of viral
infection. A subject at risk of viral infection is one who has any
risk of exposure to an infection causing pathogen. For instance, a
subject at risk may be a subject who is planning to travel to an
area where a particular type of infectious agent is found or it may
be a subject who through lifestyle or medical procedures is exposed
to bodily fluids which may contain infectious organisms or directly
to the organism or even any subject living in an area where an
infectious organism or an allergen has been identified. Subjects at
risk of developing infection also include general populations to
which a medical agency recommends vaccination with a particular
infectious organism antigen. A subject at risk of viral infection
may be identified in a variety of ways, such as by a medical
worker. Medical workers include doctors, nurses, technicians and
any other practitioners in the medical field. The subject at risk
of a viral infection may also be identified based on exposure to a
risk factor for viral infection.
[0037] In aspects of the invention the method for identifying a
risk factor for viral infection is directed at treating subjects in
anticipation of exposure to a viral agent or season (e.g., in
anticipation of the flu and cold season). Such seasonal times are
generally known and more specifically determined on an annual
basis.
[0038] A subject having an infection is a subject that has been
exposed to an infectious pathogen and has acute or chronic
detectable levels of the pathogen in the body. An infectious
disease, as used herein, is a disease arising from the presence of
a foreign microorganism in the body.
[0039] A subject at risk of developing asthma includes those
subjects that have been identified as having asthma but that don't
have the active disease during the CpG immunostimulatory
oligonucleotide treatment as well as subjects that are considered
to be at risk of developing these diseases because of genetic or
environmental factors.
[0040] Th2 cytokines, especially IL-4 and IL-5 are elevated in the
airways of asthmatic subjects. These cytokines promote important
aspects of the asthmatic inflammatory response, including IgE
isotope switching, eosinophil chemotaxis and activation and mast
cell growth. Th1 cytokines, especially IFN-.gamma. and IL-12, can
suppress the formation of Th2 clones and production of Th2
cytokines. Asthma refers to a disorder of the respiratory system
characterized by inflammation, narrowing of the airways and
increased reactivity of the airways to inhaled agents. Asthma is
frequently, although not exclusively associated with atopic or
allergic symptoms.
[0041] A subject shall mean a human or vertebrate animal including
but not limited to a dog, cat, horse, cow, pig, sheep, goat,
turkey, chicken, primate, e.g., monkey, and fish (aquaculture
species), e.g. salmon.
[0042] As used herein, the term treat, treated, or treating when
used with respect to an disorder such as an infectious disease or
asthma refers to a prophylactic treatment which increases the
resistance of a subject to development of the disease (e.g., to
infection with a pathogen) or, in other words, decreases the
likelihood that the subject will develop the disease (e.g., become
infected with the pathogen) as well as a treatment after the
subject has developed the disease in order to fight the disease
(e.g., reduce or eliminate the infection) or prevent the disease
from becoming worse.
[0043] Examples of viruses that have been found in humans include
but are not limited to: Retroviridae (e.g. human immunodeficiency
viruses, such as HIV-1 (also referred to as HDTV-III, LAVE or
HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;
Picornaviridae (e.g. polio viruses, hepatitis A virus;
enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);
Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae
(e.g. equine encephalitis viruses, rubella viruses); Flaviridae
(e.g. dengue viruses, encephalitis viruses, yellow fever viruses);
Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g. vesicular
stomatitis viruses, rabies viruses); Filoviridae (e.g. ebola
viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus,
measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.
influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga
viruses, phleboviruses and Nairo viruses); Arena viridae
(hemorrhagic fever viruses); Reoviridae (e.g. reoviruses,
orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae
(Hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae
(papilloma viruses, polyoma viruses); Adenoviridae (most
adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2,
varicella zoster virus, cytomegalovirus (CMV), herpes virus;
Poxyiridae (variola viruses, vaccinia viruses, pox viruses); and
Iridoviridae (e.g. African swine fever virus); and unclassified
viruses (e.g. the agent of delta hepatitis (thought to be a
defective satellite of hepatitis B virus), the agents of non-A,
non-B hepatitis (class 1=internally transmitted; class
2=parenterally transmitted (i.e. Hepatitis C); Norwalk and related
viruses, and astroviruses).
[0044] Both gram negative and gram positive bacteria serve as
antigens in vertebrate animals. Such gram positive bacteria
include, but are not limited to, Pasteurella species, Staphylococci
species, and Streptococcus species. Gram negative bacteria include,
but are not limited to, Escherichia coli, Pseudomonas species, and
Salmonella species. Specific examples of infectious bacteria
include but are not limited to, Helicobacter pyloris, Borelia
burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M
tuberculosis, M avium, M intracellulare, M kansaii, M gordonae),
Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria
meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group
A Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
Streptococcus (viridans group), Streptococcus faecalis,
Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus
pneumoniae, pathogenic Campylobacter sp., Enterococcus sp.,
Haemophilus influenzae, Bacillus antracis, corynebacterium
diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae,
Clostridium perfringers, Clostridium tetani, Enterobacter
aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides
sp., Fusobacterium nucleatum, Streptobacillus moniliformis,
Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia,
and Actinomyces israelli.
[0045] Examples of fungi include Cryptococcus neoformans,
Histoplasma capsulatum, Coccidioides immitis, Blastomyces
dermatitidis, Chlamydia trachomatis, Candida albicans.
[0046] Other infectious organisms (i.e., protists) include
Plasmodium spp. such as Plasmodium falciparum, Plasmodium malariae,
Plasmodium ovale, and Plasmodium vivax and Toxoplasma gondii.
Blood-borne and/or tissues parasites include Plasmodium spp.,
Babesia microti, Babesia divergens, Leishmania tropica, Leishmania
spp., Leishmania braziliensis, Leishmania donovani, Trypanosoma
gambiense and Trypanosoma rhodesiense (African sleeping sickness),
Trypanosoma cruzi (Chagas' disease), and Toxoplasma gondii.
[0047] Other medically relevant microorganisms have been described
extensively in the literature, e.g., see C. G. A Thomas, Medical
Microbiology, Bailliere Tindall, Great Britain 1983, the entire
contents of which is hereby incorporated by reference.
[0048] In some instances the viral exacerbated asthma is caused by
a respiratory virus and in particular an upper respiratory virus
such as influenza. Optionally the respiratory virus may not be RSV
(respiratory syncicial virus).
[0049] The method for treating viral exacerbated asthma may also
include the use of a combination of CpG oligonucleotides with
anti-microbials or a non-CpG asthma therapy such as an asthma
medicament. The alternative therapeutic, i.e. the anti-microbial or
asthma medicament may be administered at a different time than the
CpG oligonucleotide or at the same time as the CpG
oligonucleotide.
[0050] The asthmatic subject is administered an effective amount of
a CpG oligonucleotide for treating viral exacerbated asthma. If a
combination of therapeutics is administered the CpG oligonucleotide
may be administered to the subject in an amount effective to
prevent viral infection and the asthma medicament may be
administered to the subject when symptoms of allergy or asthma
appear. Thus, the CpG oligonucleotide may be administered to the
subject and then the asthma medicament may be subsequently
administered to the subject or they are administered together at
the same time.
[0051] The CpG oligonucleotides contain specific sequences found to
elicit an immune response. These specific sequences that elicit an
immune response are referred to as "immunostimulatory motifs", and
the oligonucleotides that contain immunostimulatory motifs are
referred to as "immunostimulatory oligonucleotide molecules" and,
equivalently, "immunostimulatory oligonucleotides". The
immunostimulatory oligonucleotides of the invention thus include at
least one immunostimulatory motif. In a preferred embodiment the
immunostimulatory motif is an "internal immunostimulatory motif".
The term "internal immunostimulatory motif" refers to the position
of the motif sequence within a longer oligonucleotide sequence,
which is longer in length than the motif sequence by at least one
nucleotide linked to both the 5' and 3' ends of the
immunostimulatory motif sequence.
[0052] The CpG oligonucleotides include at least one unmethylated
CpG dinucleotide. An oligonucleotide containing at least one
unmethylated CpG dinucleotide is a oligonucleotide molecule which
contains an unmethylated cytosine-guanine dinucleotide sequence
(i.e., "CpG DNA" or DNA containing a 5' cytosine followed by 3'
guanine and linked by a phosphate bond) and activates the immune
system. The entire CpG oligonucleotide can be unmethylated or
portions may be unmethylated but at least the C of the 5' CG 3'
must be unmethylated.
[0053] The methods of the invention may embrace the use of A class,
B class and C class CpG immunostimulatory oligonucleotides. As to
CpG oligonucleotides, it has recently been described that there are
different classes of CpG oligonucleotides. One class is potent for
activating B cells but is relatively weak in inducing IFN-.alpha.
and NK cell activation; this class has been termed the B class. The
B class CpG oligonucleotides typically are fully stabilized and
include an unmethylated CpG dinucleotide within certain preferred
base contexts. See, e.g., U.S. Pat. Nos. 6,194,388; 6,207,646;
6,214,806; 6,218,371; 6,239,116; and 6,339,068. Another class is
potent for inducing IFN-.alpha. and NK cell activation but is
relatively weak at stimulating B cells; this class has been termed
the A class. The A class CpG oligonucleotides typically have
stabilized poly-G sequences at 5' and 3' ends and a palindromic
phosphodiester CpG dinucleotide-containing sequence of at least 6
nucleotides. See, for example, published patent application
PCT/JUS00/26527 (WO 01/22990). Yet another class of CpG
oligonucleotides activates B cells and NK cells and induces
IFN-.alpha.; this class has been termed the C-class. The C-class
CpG oligonucleotides, as first characterized, typically are fully
stabilized, include a B class-type sequence and a GC-rich
palindrome or near-palindrome. This class has been described in
U.S. patent application Ser. No. 10/224,523 filed on Aug. 19, 2002,
and U.S. Ser. No. 10/978,282 filed Oct. 29, 2004 the entire
contents of which are incorporated herein by reference.
[0054] "A class" CpG immunostimulatory oligonucleotides have been
described in U.S. Non-Provisional patent application Ser. No.
09/672,126 and published PCT application PCT/US00/26527 (WO
01/22990), both filed on Sep. 27, 2000 as well as in U.S. Pat. No.
6,207,646 B 1. These oligonucleotides are characterized by the
ability to induce high levels of interferon-alpha while having
minimal effects on B cell activation. The A class CpG
immunostimulatory oligonucleotides do not necessarily contain a
hexamer palindrome GACGTC, AGCGCT, or AACGTT described by Yamamoto
and colleagues. Yamamoto S et al. J. Immunol 148:4072-6 (1992).
[0055] B class CpG immunostimulatory oligonucleotides strongly
activate human B cells but have minimal effects inducing
interferon-.alpha.. B class CpG immunostimulatory oligonucleotides
have been described in USPs 6,194,388 B1 and 6,239,116 B1, issued
on Feb. 27, 2001 and May 29, 2001 respectively.
[0056] In one embodiment the invention provides a B class CpG
oligonucleotide represented by at least the formula: 5'
X.sub.1X.sub.2CGX.sub.3X.sub.4 3' wherein X.sub.1, X.sub.2,
X.sub.3, and X.sub.4 are nucleotides. In one embodiment X.sub.2 is
adenine, guanine, or thymine. In another embodiment X.sub.3 is
cytosine, adenine, or thymine.
[0057] In another embodiment the invention provides an isolated B
class CpG oligonucleotide represented by at least the formula: 5t
N.sub.1X.sub.1X.sub.2CGX.sub.3X.sub.4N.sub.2 3' wherein X.sub.1,
X.sub.2, X.sub.3, and X.sub.4 are nucleotides and N is any
nucleotide and N.sub.1 and N.sub.2 are oligonucleotide sequences
composed of from about 0-25 N's each. In one embodiment
X.sub.1X.sub.2 is a dinucleotide selected from the group consisting
of: GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG;
and X.sub.3X.sub.4 is a dinucleotide selected from the group
consisting of: TpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA,
and CpA. Preferably X.sub.1X.sub.2 is GpA or GpT and X.sub.3X.sub.4
is TpT. In other embodiments X.sub.1 or X.sub.2 or both are purines
and X.sub.3 or X.sub.4 or both are pyrimidines or X.sub.1X.sub.2 is
GpA and X.sub.3 or X.sub.4 or both are pyrimidines. In another
preferred embodiment X.sub.1X.sub.2 is a dinucleotide selected from
the group consisting of: TpA, ApA, ApC, ApG, and GpG. In yet
another embodiment X.sub.3X.sub.4 is a dinucleotide selected from
the group consisting of: TpT, TpA, TpG, ApA, ApG, GpA, and CpA.
X.sub.1X.sub.2 in another embodiment is a dinucleotide selected
from the group consisting of: TpT, TpG, ApT, GpC, CpC, CpT, TpC,
GpT and CpG; X.sub.3 is a nucleotide selected from the group
consisting of A and T and X.sub.4 is a nucleotide, but wherein when
X.sub.1X.sub.2 is TpC, GpT, or CpG, X.sub.3X.sub.4 is not TpC, ApT
or ApC. In some embodiments the oligonucleotide has a 5'TC.
[0058] In another preferred embodiment the CpG oligonucleotide has
the sequence 5' TCN.sub.1TX.sub.1X.sub.2CGX.sub.3X.sub.4 3' (SEQ ID
NO 56). The CpG oligonucleotides of the invention in some
embodiments include X.sub.1X.sub.2 selected from the group
consisting of GpT, GpG, GpA and ApA and X.sub.3X.sub.4 is selected
from the group consisting of TpT, CpT and TpC.
[0059] The B class CpG oligonucleotide sequences of the invention
are those broadly described above as well as disclosed in PCT
Published Patent Applications PCT/US95/01570 and PCT/US97/19791,
and U.S. Pat. No. 6,194,388 B1 and U.S. Pat. No. 6,239,116 B1,
issued Feb. 27, 2001 and May 29, 2001 respectively. Exemplary
sequences include but are not limited to those disclosed in these
latter applications and patents.
[0060] The C class immunostimulatory oligonucleotides contain at
least two distinct motifs and have unique and desirable stimulatory
effects on cells of the immune system. Some of these ODN have both
a traditional "stimulatory" CpG sequence and a "GC-rich" or "B-cell
neutralizing" motif. These combination motif oligonucleotides have
immune stimulating effects that fall somewhere between those
effects associated with traditional "class B" CpG ODN, which are
strong inducers of B cell activation and dendritic cell (DC)
activation, and those effects associated with class A CpG ODN which
are strong inducers of IFN-.alpha. and natural killer (NK) cell
activation but relatively poor inducers of B-cell and DC
activation. While preferred class B CpG ODN often have
phosphorothioate backbones and preferred class A CpG ODN have mixed
or chimeric backbones, the C class of combination motif immune
stimulatory oligonucleotides may have either stabilized, e.g.,
phosphorothioate, chimeric, or phosphodiester backbones, and in
some preferred embodiments, they have semi-soft backbones.
[0061] The stimulatory domain or motif may be defined by a formula:
5' X.sub.1DCGHX.sub.2 3'. D is a nucleotide other than C. C is
cytosine. G is guanine. H is a nucleotide other than G.
[0062] X.sub.1 and X.sub.2 are any oligonucleotide sequence 0 to 10
nucleotides long. X.sub.1 may include a CG, in which case there is
preferably a T immediately preceding this CG. In some embodiments
DCG is TCG. X.sub.1 is preferably from 0 to 6 nucleotides in
length. In some embodiments X.sub.2 does not contain any poly G or
poly A motifs. In other embodiments the immunostimulatory
oligonucleotide has a poly-T sequence at the 5' end or at the 3'
end. As used herein, "poly-A" or "poly-T" shall refer to a stretch
of four or more consecutive A's or T's respectively, e.g., 5' AAAA
3' or 5' TTTT 3'. As used herein, "poly-G end" shall refer to a
stretch of four or more consecutive G's, e.g., 5' GGGG 3',
occurring at the 5' end or the 3' end of a oligonucleotide. As used
herein, "poly-G oligonucleotide" shall refer to a oligonucleotide
having the formula 5' X.sub.1X.sub.2GGGX.sub.3X.sub.4 3' wherein
X.sub.1, X.sub.2, X.sub.3, and X.sub.4 are nucleotides and
preferably at least one of X.sub.3 and X.sub.4 is a G.
[0063] Some preferred designs for the B cell stimulatory domain
under this formula comprise TTTTTCG, TCG, TTCG, TTTCG, TTTTCG,
TCGT, TTCGT, TTTCGT, TCGTCGT.
[0064] The second motif of the oligonucleotide is referred to as
either P or N and is positioned immediately 5' to X.sub.1 or
immediately 3' to X.sub.2.
[0065] N is a B-cell neutralizing sequence that begins with a CGG
trinucleotide and is at least 10 nucleotides long. A B-cell
neutralizing motif includes at least one CpG sequence in which the
CG is preceded by a C or followed by a G (Krieg A M et al. (1998)
Proc Natl Acad Sci USA 95:12631-12636) or is a CG containing DNA
sequence in which the C of the CG is methylated. As used herein,
"CpG" shall refer to a 5' cytosine (C) followed by a 3' guanine (G)
and linked by a phosphate bond. At least the C of the 5' CG 3' must
be unmethylated. Neutralizing motifs are motifs which have some
degree of immunostimulatory capability when present in an otherwise
non-stimulatory motif, but, which when present in the context of
other immunostimulatory motifs serve to reduce the
immunostimulatory potential of the other motifs.
[0066] P is a GC-rich palindrome containing sequence at least 10
nucleotides long. As used herein, "palindrome" and, equivalently,
"palindromic sequence" shall refer to an inverted repeat, i.e., a
sequence such as ABCDEE'D'C'B'A' in which A and A', B and B', etc.,
are bases capable of forming the usual Watson-Crick base pairs.
[0067] As used herein, "GC-rich palindrome" shall refer to a
palindrome having a base composition of at least two-thirds G's and
C's. In some embodiments the GC-rich domain is preferably 3' to the
"B cell stimulatory domain". In the case of a 10-base long GC-rich
palindrome, the palindrome thus contains at least 8 G's and C's. In
the case of a 12-base long GC-rich palindrome, the palindrome also
contains at least 8 G's and C's. In the case of a 14-mer GC-rich
palindrome, at least ten bases of the palindrome are G's and C's.
In some embodiments the GC-rich palindrome is made up exclusively
of G's and C's.
[0068] In some embodiments the GC-rich palindrome has a base
composition of at least 81 percent G's and C's. In the case of such
a 10-base long GC-rich palindrome, the palindrome thus is made
exclusively of G's and C's. In the case of such a 12-base long
GC-rich palindrome, it is preferred that at least ten bases (83
percent) of the palindrome are G's and C's. In some preferred
embodiments, a 12-base long GC-rich palindrome is made exclusively
of G's and C's. In the case of a 14-mer GC-rich palindrome, at
least twelve bases (86 percent) of the palindrome are G's and C's.
In some preferred embodiments, a 14-base long GC-rich palindrome is
made exclusively of G's and C's. The C's of a GC-rich palindrome
can be unmethylated or they can be methylated.
[0069] In general this domain has at least 3 Cs and Gs, more
preferably 4 of each, and most preferably 5 or more of each. The
number of Cs and Gs in this domain need not be identical. It is
preferred that the Cs and Gs are arranged so that they are able to
form a self-complementary duplex, or palindrome, such as CCGCGCGG.
This may be interrupted by As or Ts, but it is preferred that the
self-complementarity is at least partially preserved as for example
in the motifs CGACGTTCGTCG (SEQ ID NO: 49) or CGGCGCCGTGCCG (SEQ ID
NO: 50). When complementarity is not preserved, it is preferred
that the non-complementary base pairs be TG. In a preferred
embodiment there are no more than 3 consecutive bases that are not
part of the palindrome, preferably no more than 2, and most
preferably only 1. In some embodiments the GC-rich palindrome
includes at least one CGG trimer, at least one CCG trimer, or at
least one CGCG tetramer. In other embodiments the GC-rich
palindrome is not CCCCCCGGGGGG (SEQ ID NO: 51) or GGGGGGCCCCCC (SEQ
ID NO: 52), CCCCCGGGGG (SEQ ID NO: 53) or GGGGGCCCCC (SEQ ID NO:
54).
[0070] At least one of the G's of the GC rich region may be
substituted with an inosine (I). In some embodiments P includes
more than one I.
[0071] In certain embodiments the immunostimulatory oligonucleotide
has one of the following formulas 5' NX.sub.1DCGHX.sub.2 3', 5'
X.sub.1DCGHX.sub.2N 3', 5' PX.sub.1DCGHX.sub.2 3', 5'
X.sub.1DCGHX.sub.2P 3', 5' X.sub.1DCGHX.sub.2PX.sub.3 3', 5'
X.sub.1DCGHPX.sub.3 3', 5' DCGHX.sub.2PX.sub.3 3', 5'
TCGHX.sub.2PX.sub.3 3', 5' DCGHPX.sub.3 3', or 5' DCGHP 3'.
[0072] In other aspects the invention provides immune stimulatory
oligonucleotides which are defined by a formula: 5'
N.sub.1PyGN.sub.2P 3'. N.sub.1 is any sequence 1 to 6 nucleotides
long. Py is a pyrimidine. G is guanine. N.sub.2 is any sequence 0
to 30 nucleotides long. P is a GC-rich palindrome containing
sequence at least 10 nucleotides long.
[0073] N.sub.1 and N.sub.2 may contain more than 50% pyrimidines,
and more preferably more than 50% T. N.sub.1 may include a CG, in
which case there is preferably a T immediately preceding this CG.
In some embodiments N.sub.1PyG is TCG (such as ODN 5376, which has
a 5' TCGG), and most preferably a TCGN.sub.2, where N.sub.2 is not
G.
[0074] N.sub.1PyGN.sub.2P may include one or more inosine (I)
nucleotides. Either the C or the G in N1 may be replaced by
inosine, but the CpI is preferred to the IpG. For inosine
substitutions such as IpG, the optimal activity may be achieved
with the use of a "semi-soft" or chimeric backbone, where the
linkage between the IG or the CI is phosphodiester. N.sub.1 may
include at least one CI, TCI, IG or TIG motif.
[0075] In certain embodiments N.sub.1PyGN.sub.2 is a sequence
selected from the group consisting of TTTTTCG, TCG, TTCG, TTTCG,
TTTTCG, TCGT, TTCGT, TTTCGT, and TCGTCGT.
[0076] C-Class ODN are also described in U.S. patent application
Ser. No. 10/978,283 filed on Oct. 28, 2004. The nucleic acids
described therein are all incorporated by reference.
[0077] Some non limiting examples of CpG oligonucleotides useful
according to the invention include: TABLE-US-00001 SEQ ID NO
Sequence 1 T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 2
T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3
T*C_G*G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 4
T*C_G*G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 5
T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G 6
T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 7 T*C_G*A*C
G*T*T*C_G*G*C*G*C*G*C*C*G 8 T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*C*G 9
T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 10
T*C_G*T*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 11
T*C*G*T*C*G*T*T*T*T*G*A*C*G*T*T*T*T*G*T*C*G*T*T 12
T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T 13
T*C*G*T*C_G*T*T*T*T*A*C_G*G*C*G*C*C_G*T*G*C*C*G 14
T*C_G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T*T 15
T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 16
T*C_G_A_C_G_T_C_G_T_G_G*G*G*G 17
T*C*G*A*C*G*T*C*G*A*C*G*T*G*A*C*G*T*G 18
T*C*G*A*C*G*T*C*G*A*C*G*T*G*A*C*G 19
T*T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T 20
T*T*T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T 21
T*C*G*T*C_G*T*A*C_G*G*C*G*C*C_G*T*G*C*C*G 22
T*C*G*T*C_G*T*T*A*C_G*G*C*G*C*C_G*T*G*C*C*G 23
T*C*G*A*C*G*T*C*G*A*C*G*T*G*A*C*G*T*T 24
T*C*G*T*C_G*A*C_G*A*T*C_G*G*C*G*C*C_G*T*G*C*C*G 25
T*C*G*T*C*G*A*C*G*A_T_C*G*G*C*G*C*C*G*T*G*C*C*G 26
T*C*G*A*C_G*T*C*G*A*C_G*T*G*A*C*G*T*T 27
T*C*G*A*C_G*T*C*G*A*C*G*T_G*A*C*G*T*T 28
T*C*G*T*C_G*T*T*T*A*C_G*G*C*G*C*C_G*T*G*C*C*G*T 29
T*C_G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T*T 30
T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T*T 31
T*C_G*T*T*T*T*G*A*C_G*T*T 32
T*C_G*T*C_G*T*T*T_T*G*A*C_G*T*T*T*T*G*T*C_G*T*T 33
T*C_G*T*C_G*T*T*T_T*G*A*C_G*T*T*T_T*G*T*C_G*T*T 34
T*C_G*T*C_G*T_T*T_T*G_A*C_G*T_T*T_T*G_T*C_G*T*T 35
T_C_G_T_C_G_T_T_T_T_G_A_C_G_T_T_T_T_G_T_C_G_T_T 36
G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T*T 37
T*C_G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C 38
T*C_G*T*C_G*T*T*T*T*G*A*C 39 G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C 40
G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T*T 41
G*T*C_G*T*T*T*T*G*A*C_G*T*T 42 T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C
43 G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C 44 G*T*C_G*T*T*T*T*G*A*C
45 C_G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T*T 46
T*C*G*A*T*C*G*T*T*T*T_T_C_G*T*G*C*G*T*T*T*T*T 47
T*C*G*C*G*A*C_G*T*T*C*G*C*G*C_G*C*G*C*G 48
T*C_G*G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 49 CGACGTTCGTCG 50
CGGCGCCGTGCCG 51 CCCCCCGGGGGG 52 GGGGGGCCCCCC 53 CCCCCGGGGG 54
GGGGGCCCCC 55 T*G_C*T*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 56
TCXTXXCGXX 57 T*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G
[0078] The immunostimulatory oligonucleotide molecules may have a
chimeric backbone. For purposes of the instant invention, a
chimeric backbone refers to a partially stabilized backbone,
wherein at least one internucleotide linkage is phosphodiester or
phosphodiester-like, and wherein at least one other internucleotide
linkage is a stabilized internucleotide linkage, wherein the at
least one phosphodiester or phosphodiester-like linkage and the at
least one stabilized linkage are different. Since boranophosphonate
linkages have been reported to be stabilized relative to
phosphodiester linkages, for purposes of the chimeric nature of the
backbone, boranophosphonate linkages can be classified either as
phosphodiester-like or as stabilized, depending on the context. For
example, a chimeric backbone according to the instant invention
could in one embodiment include at least one phosphodiester
(phosphodiester or phosphodiester-like) linkage and at least one
boranophosphonate (stabilized) linkage. In another embodiment a
chimeric backbone according to the instant invention could include
boranophosphonate (phosphodiester or phosphodiester-like) and
phosphorothioate (stabilized) linkages. A "stabilized
internucleotide linkage" shall mean an internucleotide linkage that
is relatively resistant to in vivo degradation (e.g., via an exo-
or endo-nuclease), compared to a phosphodiester internucleotide
linkage. Preferred stabilized internucleotide linkages include,
without limitation, phosphorothioate, phosphorodithioate,
methylphosphonate, and methylphosphorothioate. Other stabilized
internucleotide linkages include, without limitation: peptide,
alkyl, dephospho, and others as described above.
[0079] Modified backbones such as phosphorothioates may be
synthesized using automated techniques employing either
phosphoramidate or H-phosphonate chemistries. Aryl- and
alkyl-phosphonates can be made, e.g., as described in U.S. Pat. No.
4,469,863; and alkylphosphotriesters (in which the charged oxygen
moiety is alkylated as described in U.S. Pat. No. 5,023,243 and
European Patent No. 092,574) can be prepared by automated solid
phase synthesis using commercially available reagents. Methods for
making other DNA backbone modifications and substitutions have been
described. Uhlmann E et al. (1990) Chem Rev 90:544; Goodchild J
(1990) Bioconjugate Chem 1:165. Methods for preparing chimeric
oligonucleotides are also known. For instance patents issued to
Uhlmann et al have described such techniques.
[0080] Mixed backbone modified ODN may be synthesized using a
commercially available DNA synthesizer and standard phosphoramidite
chemistry. (F. E. Eckstein, "Oligonucleotides and Analogues--A
Practical Approach" IRL Press, Oxford, UK, 1991, and M. D.
Matteucci and M. H. Caruthers, Tetrahedron Lett. 21, 719 (1980))
After coupling, PS linkages are introduced by sulfurization using
the Beaucage reagent (R. P. Iyer, W. Egan, J. B. Regan and S. L.
Beaucage, J. Am. Chem. Soc. 112, 1253 (1990)) (0.075 M in
acetonitrile) or phenyl acetyl disulfide (PADS) followed by capping
with acetic anhydride, 2,6-lutidine in tetrahydrofurane (1:1:8;
v:v:v) and N-methylimidazole (16% in tetrahydrofurane). This
capping step is performed after the sulfurization reaction to
minimize formation of undesired phosphodiester (PO) linkages at
positions where a phosphorothioate linkage should be located. In
the case of the introduction of a phosphodiester linkage, e.g. at a
CpG dinucleotide, the intermediate phosphorous-III is oxidized by
treatment with a solution of iodine in water/pyridine. After
cleavage from the solid support and final deprotection by treatment
with concentrated ammonia (15 hrs at 50.degree. C.), the ODN are
analyzed by HPLC on a Gen-Pak Fax column (Millipore-Waters) using a
NaCl-gradient (e.g. buffer A: 10 mM NaH.sub.2PO.sub.4 in
acetonitrile/water=1:4/v:v pH 6.8; buffer B: 10 mM
NaH.sub.2PO.sub.4, 1.5 M NaCl in acetonitrile/water=1:4/v:v; 5 to
60% B in 30 minutes at 1 ml/min) or by capillary gel
electrophoresis. The ODN can be purified by HPLC or by FPLC on a
Source High Performance column (Amersham Pharmacia).
HPLC-homogeneous fractions are combined and desalted via a C18
column or by ultrafiltration. The ODN was analyzed by MALDI-TOF
mass spectrometry to confirm the calculated mass.
[0081] The oligonucleotides of the invention can also include other
modifications. These include nonionic DNA analogs, such as alkyl-
and aryl-phosphates (in which the charged phosphonate oxygen is
replaced by an alkyl or aryl group), phosphodiester and
alkylphosphotriesters, in which the charged oxygen moiety is
alkylated. Oligonucleotides which contain diol, such as
tetraethyleneglycol or hexaethyleneglycol, at either or both
termini have also been shown to be substantially resistant to
nuclease degradation.
[0082] In some embodiments the oligonucleotides may be soft or
semi-soft oligonucleotides. A soft oligonucleotide is an
immunostimulatory oligonucleotide having a partially stabilized
backbone, in which phosphodiester or phosphodiester-like
internucleotide linkages occur only within and immediately adjacent
to at least one internal pyrimidine-purine dinucleotide (YZ).
Preferably YZ is YG, a pyrimidine-guanosine (YG) dinucleotide. The
at least one internal YZ dinucleotide itself has a phosphodiester
or phosphodiester-like internucleotide linkage. A phosphodiester or
phosphodiester-like internucleotide linkage occurring immediately
adjacent to the at least one internal YZ dinucleotide can be 5',
3', or both 5' and 3' to the at least one internal YZ
dinucleotide.
[0083] In particular, phosphodiester or phosphodiester-like
internucleotide linkages involve "internal dinucleotides". An
internal dinucleotide in general shall mean any pair of adjacent
nucleotides connected by an internucleotide linkage, in which
neither nucleotide in the pair of nucleotides is a terminal
nucleotide, i.e., neither nucleotide in the pair of nucleotides is
a nucleotide defining the 5' or 3' end of the oligonucleotide. Thus
a linear oligonucleotide that is n nucleotides long has a total of
n-1 dinucleotides and only n-3 internal dinucleotides. Each
internucleotide linkage in an internal dinucleotide is an internal
internucleotide linkage. Thus a linear oligonucleotide that is n
nucleotides long has a total of n-1 internucleotide linkages and
only n-3 internal internucleotide linkages. The strategically
placed phosphodiester or phosphodiester-like internucleotide
linkages, therefore, refer to phosphodiester or phosphodiester-like
internucleotide linkages positioned between any pair of nucleotides
in the oligonucleotide sequence. In some embodiments the
phosphodiester or phosphodiester-like internucleotide linkages are
not positioned between either pair of nucleotides closest to the 5'
or 3' end.
[0084] Preferably a phosphodiester or phosphodiester-like
internucleotide linkage occurring immediately adjacent to the at
least one internal YZ dinucleotide is itself an internal
internucleotide linkage. Thus for a sequence N.sub.1 YZ N.sub.2,
wherein N.sub.1 and N.sub.2 are each, independent of the other, any
single nucleotide, the YZ dinucleotide has a phosphodiester or
phosphodiester-like internucleotide linkage, and in addition (a)
N.sub.1 and Y are linked by a phosphodiester or phosphodiester-like
internucleotide linkage when N.sub.1 is an internal nucleotide, (b)
Z and N.sub.2 are linked by a phosphodiester or phosphodiester-like
internucleotide linkage when N.sub.2 is an internal nucleotide, or
(c) N.sub.1 and Y are linked by a phosphodiester or
phosphodiester-like internucleotide linkage when N.sub.1 is an
internal nucleotide and Z and N.sub.2 are linked by a
phosphodiester or phosphodiester-like internucleotide linkage when
N.sub.2 is an internal nucleotide.
[0085] Soft oligonucleotides according to the instant invention are
believed to be relatively susceptible to nuclease cleavage compared
to completely stabilized oligonucleotides. Without meaning to be
bound to a particular theory or mechanism, it is believed that soft
oligonucleotides of the invention are cleavable to fragments with
reduced or no immunostimulatory activity relative to full-length
soft oligonucleotides. Incorporation of at least one
nuclease-sensitive internucleotide linkage, particularly near the
middle of the oligonucleotide, is believed to provide an "off
switch" which alters the pharmacokinetics of the oligonucleotide so
as to reduce the duration of maximal immunostimulatory activity of
the oligonucleotide. This can be of particular value in tissues and
in clinical applications in which it is desirable to avoid injury
related to chronic local inflammation or immunostimulation, e.g.,
the kidney.
[0086] A semi-soft oligonucleotide is an immunostimulatory
oligonucleotide having a partially stabilized backbone, in which
phosphodiester or phosphodiester-like internucleotide linkages
occur only within at least one internal pyrimidine-purine (YZ)
dinucleotide. Semi-soft oligonucleotides generally possess
increased immunostimulatory potency relative to corresponding fully
stabilized immunostimulatory oligonucleotides. Due to the greater
potency of semi-soft oligonucleotides, semi-soft oligonucleotides
may be used, in some instances, at lower effective concentations
and have lower effective doses than conventional fully stabilized
immunostimulatory oligonucleotides in order to achieve a desired
biological effect.
[0087] It is believed that the foregoing properties of semi-soft
oligonucleotides generally increase with increasing "dose" of
phosphodiester or phosphodiester-like internucleotide linkages
involving internal YZ dinucleotides. Thus it is believed, for
example, that generally for a given oligonucleotide sequence with
five internal YZ dinucleotides, an oligonucleotide with five
internal phosphodiester or phosphodiester-like YZ internucleotide
linkages is more immunostimulatory than an oligonucleotide with
four internal phosphodiester or phosphodiester-like YG
internucleotide linkages, which in turn is more immunostimulatory
than an oligonucleotide with three internal phosphodiester or
phosphodiester-like YZ internucleotide linkages, which in turn is
more immunostimulatory than an oligonucleotide with two internal
phosphodiester or phosphodiester-like YZ internucleotide linkages,
which in turn is more immunostimulatory than an oligonucleotide
with one internal phosphodiester or phosphodiester-like YZ
internucleotide linkage. Importantly, inclusion of even one
internal phosphodiester or phosphodiester-like YZ internucleotide
linkage is believed to be advantageous over no internal
phosphodiester or phosphodiester-like YZ internucleotide linkage.
In addition to the number of phosphodiester or phosphodiester-like
internucleotide linkages, the position along the length of the
oligonucleotide can also affect potency. The soft and semi-soft
oligonucleotides will generally include, in addition to the
phosphodiester or phosphodiester-like internucleotide linkages at
preferred internal positions, 5' and 3' ends that are resistant to
degradation. Such degradation-resistant ends can involve any
suitable modification that results in an increased resistance
against exonuclease digestion over corresponding unmodified ends.
For instance, the 5' and 3' ends can be stabilized by the inclusion
there of at least one phosphate modification of the backbone. In a
preferred embodiment, the at least one phosphate modification of
the backbone at each end is independently a phosphorothioate,
phosphorodithioate, methylphosphonate, or methylphosphorothioate
internucleotide linkage. In another embodiment, the
degradation-resistant end includes one or more nucleotide units
connected by peptide or amide linkages at the 3' end.
[0088] A phosphodiester internucleotide linkage is the type of
linkage characteristic of oligonucleotides found in nature. The
phosphodiester internucleotide linkage includes a phosphorus atom
flanked by two bridging oxygen atoms and bound also by two
additional oxygen atoms, one charged and the other uncharged.
Phosphodiester internucleotide linkage is particularly preferred
when it is important to reduce the tissue half-life of the
oligonucleotide.
[0089] A phosphodiester-like internucleotide linkage is a
phosphorus-containing bridging group that is chemically and/or
diastereomerically similar to phosphodiester. Measures of
similarity to phosphodiester include susceptibility to nuclease
digestion and ability to activate RNAse H. Thus for example
phosphodiester, but not phosphorothioate, oligonucleotides are
susceptible to nuclease digestion, while both phosphodiester and
phosphorothioate oligonucleotides activate RNAse H. In a preferred
embodiment the phosphodiester-like internucleotide linkage is
boranophosphate (or equivalently, boranophosphonate) linkage. U.S.
Pat. No. 5,177,198; U.S. Pat. No. 5,859,231; U.S. Pat. No.
6,160,109; U.S. Pat. No. 6,207,819; Sergueev et al., (1998) J Am
Chem Soc 120:9417-27. In another preferred embodiment the
phosphodiester-like internucleotide linkage is diasteromerically
pure Rp phosphorothioate. It is believed that diasteromerically
pure Rp phosphorothioate is more susceptible to nuclease digestion
and is better at activating RNAse H than mixed or
diastereomerically pure Sp phosphorothioate. Stereoisomers of CpG
oligonucleotides are the subject of co-pending U.S. patent
application Ser. No. 09/361,575 filed Jul. 27, 1999, and published
PCT application PCT/US99/17100 (WO 00/06588). It is to be noted
that for purposes of the instant invention, the term
"phosphodiester-like internucleotide linkage" specifically excludes
phosphorodithioate and methylphosphonate internucleotide
linkages.
[0090] As described above the soft and semi-soft oligonucleotides
of the invention may have phosphodiester like linkages between C
and G. One example of a phosphodiester-like linkage is a
phosphorothioate linkage in an Rp conformation. Oligonucleotide
p-chirality can have apparently opposite effects on the immune
activity of a CpG oligonucleotide, depending upon the time point at
which activity is measured. At an early time point of 40 minutes,
the R.sub.p but not the S.sub.P stereoisomer of phosphorothioate
CpG oligonucleotide induces JNK phosphorylation in mouse spleen
cells. In contrast, when assayed at a late time point of 44 hr, the
S.sub.P but not the R.sub.p stereoisomer is active in stimulating
spleen cell proliferation. This difference in the kinetics and
bioactivity of the R.sub.p and S.sub.p stereoisomers does not
result from any difference in cell uptake, but rather most likely
is due to two opposing biologic roles of the p-chirality. First,
the enhanced activity of the Rp stereoisomer compared to the Sp for
stimulating immune cells at early time points indicates that the
R.sub.p may be more effective at interacting with the CpG receptor,
TLR9, or inducing the downstream signaling pathways. On the other
hand, the faster degradation of the Rp PS-oligonucleotides compared
to the Sp results in a much shorter duration of signaling, so that
the Sp PS-oligonucleotides appear to be more biologically active
when tested at later time points.
[0091] A surprisingly strong effect is achieved by the p-chirality
at the CpG dinucleotide itself. In comparison to a stereo-random
CpG oligonucleotide the congener in which the single CpG
dinucleotide was linked in Rp was slightly more active, while the
congener containing an Sp linkage was nearly inactive for inducing
spleen cell proliferation.
[0092] The size (i.e., the number of nucleotide residues along the
length of the oligonucleotide) of the immunostimulatory
oligonucleotide may also contribute to the stimulatory activity of
the oligonucleotide. For facilitating uptake into cells
immunostimulatory oligonucleotides preferably have a minimum length
of 6 nucleotide residues. Oligonucleotides of any size greater than
6 nucleotides (even many kb long) are capable of inducing an immune
response according to the invention if sufficient immunostimulatory
motifs are present, since larger oligonucleotides are degraded
inside of cells. It is believed by the instant inventors that
semi-soft oligonucleotides as short as 4 nucleotides can also be
immunostimulatory if they can be delivered to the interior of the
cell. In certain preferred embodiments according to the instant
invention, the immunostimulatory oligonucleotides are between 4 and
100 nucleotides long. In typical embodiments the immunostimulatory
oligonucleotides are between 6 and 40 nucleotides long. In certain
embodiments according to the instant invention, the
immunostimulatory oligonucleotides are between 6 and 19 nucleotides
long. The immunostimulatory oligonucleotides generally have a
length in the range of between 4 and 100 and in some embodiments 8
and 40. The length may be in the range of between 16 and 24
nucleotides.
[0093] The term "oligonucleotide" also encompasses oligonucleotides
with substitutions or modifications, such as in the bases and/or
sugars. For example, they include oligonucleotides having backbone
sugars that are covalently attached to low molecular weight organic
groups other than a hydroxyl group at the 2' position and other
than a phosphate group or hydroxy group at the 5' position. Thus
modified oligonucleotides may include a 2'-O-alkylated ribose
group. In addition, modified oligonucleotides may include sugars
such as arabinose or 2'-fluoroarabinose instead of ribose. Thus the
oligonucleotides may be heterogeneous in backbone composition
thereby containing any possible combination of polymer units linked
together such as peptide-nucleic acids (which have an amino acid
backbone with oligonucleotide bases).
[0094] Oligonucleotides also include substituted purines and
pyrimidines such as C-5 propyne pyrimidine and
7-deaza-7-substituted purine modified bases. Wagner R W et al.
(1996) Nat Biotechnol 14:840-4. Purines and pyrimidines include but
are not limited to adenine, cytosine, guanine, thymine,
5-methylcytosine, 5-hydroxycytosine, 5-fluorocytosine,
2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine,
hypoxanthine, and other naturally and non-naturally occurring
nucleobases, substituted and unsubstituted aromatic moieties. Other
such modifications are well known to those of skill in the art.
[0095] The immunostimulatory oligonucleotides of the instant
invention can encompass various chemical modifications and
substitutions, in comparison to natural RNA and DNA, involving a
phosphodiester internucleotide bridge, a .beta.-D-ribose unit
and/or a natural nucleotide base (adenine, guanine, cytosine,
thymine, uracil). Examples of chemical modifications are known to
the skilled person and are described, for example, in Uhlmann E et
al. (1990) Chem Rev 90:543; "Protocols for Oligonucleotides and
Analogs" Synthesis and Properties & Synthesis and Analytical
Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA 1993; Crooke
ST et al. (1996) Annu Rev Pharmacol Toxicol 36:107-129; and
Hunziker J et al. (1995) Mod Synth Methods 7:331-417. An
oligonucleotide according to the invention may have one or more
modifications, wherein each modification is located at a particular
phosphodiester internucleotide bridge and/or at a particular
.beta.-D-ribose unit and/or at a particular natural nucleotide base
position in comparison to an oligonucleotide of the same sequence
which is composed of natural DNA or RNA.
[0096] For example, the invention relates to an oligonucleotide
which may comprise one or more modifications and wherein each
modification is independently selected from: [0097] a) the
replacement of a phosphodiester internucleotide bridge located at
the 3' and/or the 5' end of a nucleotide by a modified
internucleotide bridge, [0098] b) the replacement of phosphodiester
bridge located at the 3' and/or the 5' end of a nucleotide by a
dephospho bridge, [0099] c) the replacement of a sugar phosphate
unit from the sugar phosphate backbone by another unit, [0100] d)
the replacement of a .beta.-D-ribose unit by a modified sugar unit,
and [0101] e) the replacement of a natural nucleotide base by a
modified nucleotide base.
[0102] More detailed examples for the chemical modification of an
oligonucleotide are as follows.
[0103] A phosphodiester internucleotide bridge located at the 3'
and/or the 5' end of a nucleotide can be replaced by a modified
internucleotide bridge, wherein the modified internucleotide bridge
is for example selected from phosphorothioate, phosphorodithioate,
NR.sup.1R.sup.2-phosphoramidate, boranophosphate,
.alpha.-hydroxybenzyl phosphonate,
phosphate-(C.sub.1-C.sub.21)-O-alkyl ester,
phosphate-[(C.sub.6-C.sub.12)aryl-(C.sub.1-C.sub.21)-O-alkyl]ester,
(C.sub.1-C.sub.8)alkylphosphonate and/or
(C.sub.6-C.sub.12)arylphosphonate bridges,
(C.sub.7-C.sub.12)-.alpha.-hydroxymethyl-aryl (e.g., disclosed in
WO 95/01363), wherein (C.sub.6-C.sub.12)aryl,
(C.sub.6-C.sub.20)aryl and (C.sub.6-C.sub.14)aryl are optionally
substituted by halogen, alkyl, alkoxy, nitro, cyano, and where
R.sup.1 and R.sup.2 are, independently of each other, hydrogen,
(C.sub.1-C.sub.18)-alkyl, (C.sub.6-C.sub.20)-aryl,
(C.sub.6-C.sub.14)-aryl-(C.sub.1-C.sub.8)-alkyl, preferably
hydrogen, (C.sub.1-C.sub.8)-alkyl, preferably
(C.sub.1-C.sub.4)-alkyl and/or methoxyethyl, or R.sup.1 and R.sup.2
form, together with the nitrogen atom carrying them, a 5-6-membered
heterocyclic ring which can additionally contain a further
heteroatom from the group O, S and N.
[0104] The replacement of a phosphodiester bridge located at the 3'
and/or the 5' end of a nucleotide by a dephospho bridge (dephospho
bridges are described, for example, in Uhlmann E and Peyman A in
"Methods in Molecular Biology", Vol. 20, "Protocols for
Oligonucleotides and Analogs", S. Agrawal, Ed., Humana Press,
Totowa 1993, Chapter 16, pp. 355 ff), wherein a dephospho bridge is
for example selected from the dephospho bridges formacetal,
3'-thioformacetal, methylhydroxylamine, oxime,
methylenedimethyl-hydrazo, dimethylenesulfone and/or silyl
groups.
[0105] A sugar phosphate unit (i.e., a .beta.-D-ribose and
phosphodiester internucleotide bridge together forming a sugar
phosphate unit) from the sugar phosphate backbone (i.e., a sugar
phosphate backbone is composed of sugar phosphate units) can be
replaced by another unit, wherein the other unit is for example
suitable to build up a "morpholino-derivative" oligomer (as
described, for example, in Stirchak E P et al. (1989)
Oligonucleotides Res 17:6129-41), that is, e.g., the replacement by
a morpholino-derivative unit; or to build up a polyamide
oligonucleotide ("PNA"; as described for example, in Nielsen P E et
al. (1994) Bioconjug Chem 5:3-7), that is, e.g., the replacement by
a PNA backbone unit, e.g., by 2-aminoethylglycine.
[0106] A .beta.-ribose unit or a .beta.-D-2'-deoxyribose unit can
be replaced by a modified sugar unit, wherein the modified sugar
unit is for example selected from .beta.-D-ribose,
.alpha.-D-2'-deoxyribose, L-2'-deoxyribose, 2'-F-2'-deoxyribose,
2'-F-arabinose, 2'-O--(C.sub.1-C.sub.6)alkyl-ribose, preferably
2'-O--(C.sub.1-C.sub.6)alkyl-ribose is 2'-O-methylribose,
2'-O--(C.sub.2-C.sub.6)alkenyl-ribose,
2'-[O--(C.sub.1-C.sub.6)alkyl-O--(C.sub.1-C.sub.6)alkyl]-ribose,
2'-NH.sub.2-2'-deoxyribose, .beta.-D-xylo-furanose,
.alpha.-arabinofuranose,
2,4-dideoxy-.beta.-D-erythro-hexo-pyranose, and carbocyclic
(described, for example, in Froehler J (1992) Am Chem Soc 114:8320)
and/or open-chain sugar analogs (described, for example, in
Vandendriessche et al. (1993) Tetrahedron 49:7223) and/or
bicyclosugar analogs (described, for example, in Tarkov M et al.
(1993) Helv Chim Acta 76:481).
[0107] In some embodiments the sugar is 2'-O-methylribose,
particularly for one or both nucleotides linked by a phosphodiester
or phosphodiester-like internucleotide linkage.
[0108] Oligonucleotides also include substituted purines and
pyrimidines such as C-5 propyne pyrimidine and
7-deaza-7-substituted purine modified bases. Wagner R W et al.
(1996) Nat Biotechnol 14:840-4. Purines and pyrimidines include but
are not limited to adenine, cytosine, guanine, and thymine, and
other naturally and non-naturally occurring nucleobases,
substituted and unsubstituted aromatic moieties.
[0109] A modified base is any base which is chemically distinct
from the naturally occurring bases typically found in DNA and RNA
such as T, C, G, A, and U, but which share basic chemical
structures with these naturally occurring bases. The modified
nucleotide base may be, for example, selected from hypoxanthine,
uracil, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil,
5-aminouracil, 5-(C.sub.1-C.sub.6)-alkyluracil,
5-(C.sub.2-C.sub.6)-alkenyluracil,
5-(C.sub.2-C.sub.6)-alkynyluracil, 5-(hydroxymethyl)uracil,
5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine,
5-(C.sub.1-C.sub.6)-alkylcytosine,
5-(C.sub.2-C.sub.6)-alkenylcytosine,
5-(C.sub.2-C.sub.6)-alkynylcytosine, 5-chlorocytosine,
5-fluorocytosine, 5-bromocytosine, N.sup.2-dimethylguanine,
2,4-diamino-purine, 8-azapurine, a substituted 7-deazapurine,
preferably 7-deaza-7-substituted and/or 7-deaza-8-substituted
purine, 5-hydroxymethylcytosine, N4-alkylcytosine, e.g.,
N4-ethylcytosine, 5-hydroxydeoxycytidine,
5-hydroxymethyldeoxycytidine, N4-alkyldeoxycytidine, e.g.,
N4-ethyldeoxycytidine, 6-thiodeoxyguanosine, and
deoxyribonucleotides of nitropyrrole, C5-propynylpyrimidine, and
diaminopurine e.g., 2,6-diaminopurine, inosine, 5-methylcytosine,
2-aminopurine, 2-amino-6-chloropurine, hypoxanthine or other
modifications of a natural nucleotide bases. This list is meant to
be exemplary and is not to be interpreted to be limiting.
[0110] In particular formulas described herein a set of modified
bases is defined. For instance the letter Y is used to refer to a
nucleotide containing a cytosine or a modified cytosine. A modified
cytosine as used herein is a naturally occurring or non-naturally
occurring pyrimidine base analog of cytosine which can replace this
base without impairing the immunostimulatory activity of the
oligonucleotide. Modified cytosines include but are not limited to
5-substituted cytosines (e.g. 5-methyl-cytosine, 5-fluoro-cytosine,
5-chloro-cytosine, 5-bromo-cytosine, 5-iodo-cytosine,
5-hydroxy-cytosine, 5-hydroxymethyl-cytosine,
5-difluoromethyl-cytosine, and unsubstituted or substituted
5-alkynyl-cytosine), 6-substituted cytosines, N4-substituted
cytosines (e.g. N4-ethyl-cytosine), 5-aza-cytosine,
2-mercapto-cytosine, isocytosine, pseudo-isocytosine, cytosine
analogs with condensed ring systems (e.g. N,N'-propylene cytosine
or phenoxazine), and uracil and its derivatives (e.g.
5-fluoro-uracil, 5-bromo-uracil, 5-bromovinyl-uracil,
4-thio-uracil, 5-hydroxy-uracil, 5-propynyl-uracil). Some of the
preferred cytosines include 5-methyl-cytosine, 5-fluoro-cytosine,
5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, and
N4-ethyl-cytosine. In another embodiment of the invention, the
cytosine base is substituted by a universal base (e.g.
3-nitropyrrole, P-base), an aromatic ring system (e.g.
fluorobenzene or difluorobenzene) or a hydrogen atom (dSpacer).
[0111] The letter Z is used to refer to guanine or a modified
guanine base. A modified guanine as used herein is a naturally
occurring or non-naturally occurring purine base analog of guanine
which can replace this base without impairing the immunostimulatory
activity of the oligonucleotide. Modified guanines include but are
not limited to 7-deazaguanine, 7-deaza-7-substituted guanine (such
as 7-deaza-7-(C2-C6)alkynylguanine), 7-deaza-8-substituted guanine,
hypoxanthine, N2-substituted guanines (e.g. N2-methyl-guanine),
5-amino-3-methyl-3H,6H-thiazolo [4,5-d]pyrimidine-2,7-dione,
2,6-diaminopurine, 2-aminopurine, purine, indole, adenine,
substituted adenines (e.g. N6-methyl-adenine, 8-oxo-adenine)
8-substituted guanine (e.g. 8-hydroxyguanine and 8-bromoguanine),
and 6-thioguanine. In another embodiment of the invention, the
guanine base is substituted by a universal base (e.g.
4-methyl-indole, 5-nitro-indole, and K-base), an aromatic ring
system (e.g. benzimidazole or dichloro-benzimidazole,
1-methyl-1H-[1,2,4]triazole-3-carboxylic acid amide) or a hydrogen
atom (dSpacer).
[0112] The oligonucleotides may have one or more accessible 5'
ends. It is possible to create modified oligonucleotides having two
such 5' ends. This may be achieved, for instance by attaching two
oligonucleotides through a 3'-3' linkage to generate an
oligonucleotide having one or two accessible 5' ends. The
3'-3'-linkage may be a phosphodiester, phosphorothioate or any
other modified internucleotide bridge. Methods for accomplishing
such linkages are known in the art. For instance, such linkages
have been described in Seliger, H.; et al., Oligonucleotide analogs
with terminal 3'-3'- and 5'-5'-internucleotidic linkages as
antisense inhibitors of viral gene expression, Nucleotides &
Nucleotides (1991), 10(1-3), 469-77 and Jiang, et al.,
Pseudo-cyclic oligonucleotides: in vitro and in vivo properties,
Bioorganic & Medicinal Chemistry (1999), 7(12), 2727-2735.
[0113] Additionally, 3'-3'-linked oligonucleotides where the
linkage between the 3'-terminal nucleotides is not a
phosphodiester, phosphorothioate or other modified bridge, can be
prepared using an additional spacer, such as tri- or
tetra-ethylenglycol phosphate moiety (Durand, M. et al,
Triple-helix formation by an oligonucleotide containing one (dA) 12
and two (dT) 12 sequences bridged by two hexaethylene glycol
chains, Biochemistry (1992), 31(38), 9197-204, U.S. Pat. No.
5,658,738, and U.S. Pat. No. 5,668,265). Alternatively, the
non-nucleotidic linker may be derived from ethanediol, propanediol,
or from an abasic deoxyribose (dSpacer) unit (Fontanel, Marie
Laurence et al., Sterical recognition by T4 polynucleotide kinase
of non-nucleosidic moieties 5'-attached to oligonucleotides;
Oligonucleotides Research (1994), 22(11), 2022-7) using standard
phosphoramidite chemistry. The non-nucleotidic linkers can be
incorporated once or multiple times, or combined with each other
allowing for any desirable distance between the 3'-ends of the two
ODNs to be linked.
[0114] The oligonucleotides are partially resistant to degradation
(e.g., are stabilized). A "stabilized oligonucleotide molecule"
shall mean an oligonucleotide that is relatively resistant to in
vivo degradation (e.g. via an exo- or endo-nuclease).
Oligonucleotide stabilization can be accomplished via backbone
modifications. Oligonucleotides having phosphorothioate linkages
provide maximal activity and protect the oligonucleotide from
degradation by intracellular exo- and endo-nucleases. Other
modified oligonucleotides include phosphodiester modified
oligonucleotides, combinations of phosphodiester and
phosphorothioate oligonucleotide, methylphosphonate,
methylphosphorothioate, phosphorodithioate, p-ethoxy, and
combinations thereof.
[0115] Modified backbones such as phosphorothioates may be
synthesized using automated techniques employing either
phosphoramidate or H-phosphonate chemistries. Aryl-and
alkyl-phosphonates can be made, e.g., as described in U.S. Pat. No.
4,469,863; and alkylphosphotriesters (in which the charged oxygen
moiety is alkylated as described in U.S. Pat. No. 5,023,243 and
European Patent No. 092,574) can be prepared by automated solid
phase synthesis using commercially available reagents. Methods for
making other DNA backbone modifications and substitutions have been
described (e.g., Uhlmann, E. and Peyman, A., Chem. Rev. 90:544,
1990; Goodchild, J., Bioconjugate Chem. 1:165, 1990).
[0116] Other stabilized oligonucleotides include: nonionic DNA
analogs, such as alkyl- and aryl-phosphates (in which the charged
phosphonate oxygen is replaced by an alkyl or aryl group),
phosphodiester and alkylphosphotriesters, in which the charged
oxygen moiety is alkylated. Oligonucleotides which contain diol,
such as tetraethyleneglycol or hexaethyleneglycol, at either or
both termini have also been shown to be substantially resistant to
nuclease degradation.
[0117] The immunostimulatory oligonucleotides may also contain one
or more unusual linkages between the nucleotide or
nucleotide-analogous moieties. The usual internucleoside linkage is
a 3'5'-linkage. All other linkages are considered to be unusual
internucleoside linkages, such as 2'5'-, 5'5'-, 3'3'-, 2'2'-,
2'3'-linkages. The nomenclature 2' to 5' is chosen according to the
carbon atom of ribose. However, if unnatural sugar moieties are
employed, such as ring-expanded sugar analogs (e.g. hexanose,
cylohexene or pyranose) or bi- or tricyclic sugar analogs, then
this nomenclature changes according to the nomenclature of the
monomer. In 3'-deoxy-.beta.-D-ribopyranose analogs (also called
p-DNA), the mononucleotides are e.g. connected via a
4'2'-linkage.
[0118] If the oligonucleotide contains one 3'3'-linkage, then this
oligonucleotide may have two unlinked 5'-ends. Similarly, if the
oligonucleotide contains one 5'5'-linkage, then this
oligonucleotide may have two unlinked 3'-ends. The accessibility of
unlinked ends of nucleotides may be better accessible by their
receptors. Both types of unusual linkages (3'3'- and 5'5') were
described by Ramalho Ortigao et al. (Antisense Research and
Development (1992) 2, 129-46), whereby oligonucleotides having a
3'3'-linkage were reported to show enhanced stability towards
cleavage by nucleases.
[0119] Different types of linkages can also be combined in one
molecule which may lead to branching of the oligomer. If one part
of the oligonucleotide is connected at the 3'-end via a
3'3'-linkage to a second oligonucleotide part and at the 2'-end via
a 2'3'-linkage to a third part of the molecule, this results e.g.
in a branched oligonucleotide with three 5'-ends (3'3'-,
2'3'-branched).
[0120] In principle, linkages between different parts of an
oligonucleotide or between different oligonucleotides,
respectively, can occur via all parts of the molecule, as long as
this does not negatively interfere with the recognition by its
receptor. According to the nature of the oligonucleotide, the
linkage can involve the sugar moiety (Su), the heterocyclic
nucleobase (Ba) or the phosphate backbone (Ph). Thus, linkages of
the type Su-Su, Su-Ph, Su-Ba, Ba-Ba, Ba-Su, Ba-Ph, Ph-Ph, Ph-Su,
and Ph-Ba are possible. If the oligonucleotides are further
modified by certain non-nucleotidic substituents, the linkage can
also occur via the modified parts of the oligonucleotides. These
modifications also include modified oligonucleotides, e.g. PNA,
LNA, or Morpholino Oligonucleotide analogs.
[0121] The linkages are preferably composed of C, H, N, O, S, B, P,
and Halogen, containing 3 to 300 atoms. An example with 3 atoms is
an acetal linkage (ODN1-3'-O--CH.sub.2--O-3'-ODN2) connecting e.g.
the 3'-hydroxy group of one nucleotide to the 3'-hydroxy group of a
second oligonucleotide. An example with about 300 atoms is PEG-40
(tetraconta polyethyleneglycol). Preferred linkages are
phosphodiester, phosphorothioate, methylphosphonate,
phosphoramidate, boranophosphonate, amide, ether, thioether,
acetal, thioacetal, urea, thiourea, sulfonamide, Schiff Base and
disulfide linkages. It is also possible to use the Solulink
BioConjugation System i.e., (www.trilinkbiotech.com).
[0122] If the oligonucleotide is composed of two or more sequence
parts, these parts can be identical or different. Thus, in an
oligonucleotide with a 3'3'-linkage, the sequences can be identical
5'-ODN1-3'3'-ODN1-5' or different 5'-ODN1-3'3'-ODN2-5'.
Furthermore, the chemical modification of the various
oligonucleotide parts as well as the linker connecting them may be
different. Since the uptake of short oligonucleotides appears to be
less efficient than that of long oligonucleotides, linking of two
or more short sequences results in improved immune stimulation. The
length of the short oligonucleotides is preferably 2-20
nucleotides, more preferably 3-16 nucleotides, but most preferably
5-10 nucleotides. Preferred are linked oligonucleotides which have
two or more unlinked 5'-ends.
[0123] The oligonucleotide partial sequences may also be linked by
non-nucleotidic linkers, in particular abasic linkers (dSpacers),
trietyhlene glycol units or hexaethylene glycol units. Further
preferred linkers are alkylamino linkers, such as C3, C6, C12
aminolinkers, and also alkylthiol linkers, such as C3 or C6 thiol
linkers. The oligonucleotides can also be linked by aromatic
residues which may be further substituted by alkyl or substituted
alkyl groups. The oligonucleotides may also contain a Doubler or
Trebler unit (www.glenres.com), in particular those
oligonucleotides with a 3'3'-linkage. Branching of the
oligonucleotides by multiple doubler, trebler, or other multiplier
units leads to dendrimers which are a further embodiment of this
invention. The oligonucleotides may also contain linker units
resulting from peptide modifying reagents or oligonucleotide
modifying reagents (www.glenres.com). Furthermore, it may contain
one or more natural or unnatural amino acid residues which are
connected by peptide (amide) linkages.
[0124] Another possibility for linking oligonucleotides is via
crosslinking of the heterocyclic bases (Verma and Eckstein; Annu.
Rev. Biochem. (1998) 67: 99-134; page 124). A linkage between the
sugar moiety of one sequence part with the heterocyclic base of
another sequence part (Iyer et al. Curr. Opin. Mol. Therapeutics
(1999) 1: 344-358; page 352) may also be used.
[0125] The different oligonucleotides are synthesized by
established methods and can be linked together on-line during
solid-phase synthesis. Alternatively, they may be linked together
post-synthesis of the individual partial sequences. ##STR1##
##STR2##
[0126] CpG immunostimulatory oligonucleotides can be combined with
other therapeutic agents. The CpG immunostimulatory oligonucleotide
and other therapeutic agent may be administered simultaneously or
sequentially. When the other therapeutic agents are administered
simultaneously they can be administered in the same or separate
formulations, but are administered at the same time. The other
therapeutic agents are administered sequentially with one another
and with CpG immunostimulatory oligonucleotide, when the
administration of the other therapeutic agents and the CpG
immunostimulatory oligonucleotide is temporally separated. The
separation in time between the administration of these compounds
may be a matter of minutes or it may be longer. Other therapeutic
agents include but are not limited to anti-microbials and anti
asthma medicaments.
[0127] The oligonucleotides of the invention may be administered to
a subject with an anti-microbial agent. An anti-microbial agent, as
used herein, refers to a naturally-occurring or synthetic compound
which is capable of killing or inhibiting infectious
microorganisms. The type of anti-microbial agent useful according
to the invention will depend upon the type of microorganism with
which the subject is infected or at risk of becoming infected.
Anti-microbial agents include but are not limited to anti-bacterial
agents, anti-viral agents, anti-fungal agents and anti-parasitic
agents. Phrases such as "anti-infective agent", "anti-bacterial
agent", "anti-viral agent", "anti-fungal agent", "anti-parasitic
agent" and "parasiticide" have well-established meanings to those
of ordinary skill in the art and are defined in standard medical
texts. Briefly, anti-bacterial agents kill or inhibit bacteria, and
include antibiotics as well as other synthetic or natural compounds
having similar functions. Antibiotics are low molecular weight
molecules which are produced as secondary metabolites by cells,
such as microorganisms. In general, antibiotics interfere with one
or more bacterial functions or structures which are specific for
the microorganism and which are not present in host cells.
Anti-viral agents can be isolated from natural sources or
synthesized and are useful for killing or inhibiting viruses.
Anti-fungal agents are used to treat superficial fungal infections
as well as opportunistic and primary systemic fungal infections.
Anti-parasite agents kill or inhibit parasites.
[0128] Examples of anti-parasitic agents, also referred to as
parasiticides useful for human administration include but are not
limited to albendazole, amphotericin B, benznidazole, bithionol,
chloroquine HCl, chloroquine phosphate, clindamycin,
dehydroemetine, diethylcarbamazine, diloxamide furoate,
eflomithine, furazolidaone, glucocorticoids, halofantrine,
iodoquinol, ivermectin, mebendazole, mefloquine, meglumine
antimoniate, melarsoprol, metrifonate, metronidazole, niclosamide,
nifurtimox, oxamniquine, paromomycin, pentamidine isethionate,
piperazine, praziquantel, primaquine phosphate, proguanil, pyrantel
pamoate, pyrimethanmine-sulfonamides, pyrimethanmine-sulfadoxine,
quinacrine HCl, quinine sulfate, quinidine gluconate, spiramycin,
stibogluconate sodium (sodium antimony gluconate), suramin,
tetracycline, doxycycline, thiabendazole, timidazole,
trimethroprim-sulfamethoxazole, and tryparsamide some of which are
used alone or in combination with others.
[0129] Antibacterial agents kill or inhibit the growth or function
of bacteria. A large class of antibacterial agents is antibiotics.
Antibiotics, which are effective for killing or inhibiting a wide
range of bacteria, are referred to as broad spectrum antibiotics.
Other types of antibiotics are predominantly effective against the
bacteria of the class gram-positive or gram-negative. These types
of antibiotics are referred to as narrow spectrum antibiotics.
Other antibiotics which are effective against a single organism or
disease and not against other types of bacteria, are referred to as
limited spectrum antibiotics. Antibacterial agents are sometimes
classified based on their primary mode of action. In general,
antibacterial agents are cell wall synthesis inhibitors, cell
membrane inhibitors, protein synthesis inhibitors, oligonucleotide
synthesis or functional inhibitors, and competitive inhibitors.
[0130] Antiviral agents are compounds which prevent infection of
cells by viruses or replication of the virus within the cell. There
are many fewer antiviral drugs than antibacterial drugs because the
process of viral replication is so closely related to DNA
replication within the host cell, that non-specific antiviral
agents would often be toxic to the host. There are several stages
within the process of viral infection which can be blocked or
inhibited by antiviral agents. These stages include, attachment of
the virus to the host cell (immunoglobulin or binding peptides),
uncoating of the virus (e.g. amantadine), synthesis or translation
of viral mRNA (e.g. interferon), replication of viral RNA or DNA
(e.g. nucleotide analogues), maturation of new virus proteins (e.g.
protease inhibitors), and budding and release of the virus.
[0131] Nucleotide analogues are synthetic compounds which are
similar to nucleotides, but which have an incomplete or abnormal
deoxyribose or ribose group. Once the nucleotide analogues are in
the cell, they are phosphorylated, producing the triphosphate
formed which competes with normal nucleotides for incorporation
into the viral DNA or RNA. Once the triphosphate form of the
nucleotide analogue is incorporated into the growing
oligonucleotide chain, it causes irreversible association with the
viral polymerase and thus chain termination. Nucleotide analogues
include, but are not limited to, acyclovir (used for the treatment
of herpes simplex virus and varicella-zoster virus), gancyclovir
(useful for the treatment of cytomegalovirus), idoxuridine,
ribavirin (useful for the treatment of respiratory syncitial
virus), dideoxyinosine, dideoxycytidine, zidovudine
(azidothymidine), imiquimod, and resimiquimod.
[0132] The interferons are cytokines which are secreted by
virus-infected cells as well as immune cells. The interferons
function by binding to specific receptors on cells adjacent to the
infected cells, causing the change in the cell which protects it
from infection by the virus. .alpha. and .beta.-interferon also
induce the expression of Class I and Class II MHC molecules on the
surface of infected cells, resulting in increased antigen
presentation for host immune cell recognition. .alpha. and
.beta.-interferons are available as recombinant forms and have been
used for the treatment of chronic hepatitis B and C infection. At
the dosages which are effective for anti-viral therapy, interferons
have severe side effects such as fever, malaise and weight
loss.
[0133] Anti-viral agents useful in the invention include but are
not limited to immunoglobulins, amantadine, interferons, nucleotide
analogues, and protease inhibitors. Specific examples of
anti-virals include but are not limited to Acemannan; Acyclovir;
Acyclovir Sodium; Adefovir; Alovudine; Alvircept Sudotox;
Amantadine Hydrochloride; Aranotin; Arildone; Atevirdine Mesylate;
Avridine; Cidofovir; Cipamfylline; Cytarabine Hydrochloride;
Delavirdine Mesylate; Desciclovir; Didanosine; Disoxaril;
Edoxudine; Enviradene; Enviroxime; Famciclovir; Famotine
Hydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscarnet
Sodium; Fosfonet Sodium; Ganciclovir; Ganciclovir Sodium;
Idoxuridine; Kethoxal; Lamivudine; Lobucavir; Memotine
Hydrochloride; Methisazone; Nevirapine; Penciclovir; Pirodavir;
Ribavirin; Rimantadine Hydrochloride; Saquinavir Mesylate;
Somantadine Hydrochloride; Sorivudine; Statolon; Stavudine;
Tilorone Hydrochloride; Trifluridine; Valacyclovir Hydrochloride;
Vidarabine; Vidarabine Phosphate; Vidarabine Sodium Phosphate;
Viroxime; Zalcitabine; Zidovudine; and Zinviroxime.
[0134] Anti-fungal agents are useful for the treatment and
prevention of infective fungi. Anti-fungal agents are sometimes
classified by their mechanism of action. Some anti-fungal agents
function as cell wall inhibitors by inhibiting glucose synthase.
These include, but are not limited to, basiungin/ECB. Other
anti-fungal agents function by destabilizing membrane integrity.
These include, but are not limited to, immidazoles, such as
clotrimazole, sertaconzole, fluconazole, itraconazole,
ketoconazole, miconazole, and voriconacole, as well as FK 463,
amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292,
butenafine, and terbinafine. Other anti-fungal agents function by
breaking down chitin (e.g. chitinase) or immunosuppression (501
cream).
[0135] An "asthma medicament" as used herein is a composition of
matter which reduces the symptoms, inhibits the asthmatic reaction,
or prevents the development of an asthmatic reaction. Various types
of medicaments for the treatment of asthma are described in the
Guidelines For The Diagnosis and Management of Asthma, Expert Panel
Report 2, NIH Publication No. 97/4051, Jul. 19, 1997, the entire
contents of which are incorporated herein by reference. The summary
of the medicaments as described in the NIH publication is presented
below.
[0136] Asthma medicaments include, but are not limited to,
steroids, PDE-4 inhibitors, bronchodilator/beta-2 agonists, K+
channel openers, VLA-4 antagonists, neurokin antagonists, TXA2
synthesis inhibitors, xanthanines, arachidonic acid antagonists, 5
lipoxygenase inhibitors, thromboxin A2 receptor antagonists,
thromboxane A2 antagonists, inhibitor of 5-lipox activation
proteins, and protease inhibitors.
[0137] Bronchodilator/beta-2 agonists are a class of compounds
which cause bronchodilation or smooth muscle relaxation.
Bronchodilator/beta-2 agonists include, but are not limited to,
salmeterol, salbutamol, albuterol, terbutaline, D2522/formoterol,
fenoterol, bitolterol, pirbuerol methylxanthines and orciprenaline.
Long-acting .beta..sub.2 agonists and bronchodilators are compounds
which are used for long-term prevention of symptoms in addition to
the anti-inflammatory therapies. They function by causing
bronchodilation, or smooth muscle relaxation, following adenylate
cyclase activation and increase in cyclic AMP producing functional
antagonism of bronchoconstriction. These compounds also inhibit
mast cell mediator release, decrease vascular permeability and
increase mucociliary clearance. Long-acting .beta.2 agonists
include, but are not limited to, salmeterol and albuterol. These
compounds are usually used in combination with corticosteroids and
generally are not used without any inflammatory therapy. They have
been associated with side effects such as tachycardia, skeletal
muscle tremor, hypokalemia, and prolongation of QTc interval in
overdose.
[0138] Methylxanthines, including for instance theophylline, have
been used for long-term control and prevention of symptoms. These
compounds cause bronchodilation resulting from phosphodiesterase
inhibition and likely adenosine antagonism. It is also believed
that these compounds may effect eosinophilic infiltration into
bronchial mucosa and decrease T-lymphocyte numbers in the
epithelium. Dose-related acute toxicities are a particular problem
with these types of compounds. As a result, routine serum
concentration must be monitored in order to account for the
toxicity and narrow therapeutic range arising from individual
differences in metabolic clearance. Side effects include
tachycardia, nausea and vomiting, tachyarrhythmias, central nervous
system stimulation, headache, seizures, hematemesis, hyperglycemia
and hypokalemia. Short-acting .beta..sub.2 agonists/bronchodilators
relax airway smooth muscle, causing the increase in air flow. These
types of compounds are a preferred drug for the treatment of acute
asthmatic systems. Previously, short-acting .beta..sub.2 agonists
had been prescribed on a regularly-scheduled basis in order to
improve overall asthma symptoms. Later reports, however, suggested
that regular use of this class of drugs produced significant
diminution in asthma control and pulmonary function (Sears, et al.
Lancet; 336:1391-6, 1990). Other studies showed that regular use of
some types of .beta..sub.2 agonists produced no harmful effects
over a four-month period but also produced no demonstrable effects
(Drazen, et al., N. Eng. J. Med.; 335:841-7, 1996). As a result of
these studies, the daily use of short-acting .beta..sub.2 agonists
is not generally recommended. Short-acting .beta..sub.2 agonists
include, but are not limited to, albuterol, bitolterol, pirbuterol,
and terbutaline. Some of the adverse effects associated with the
mastration of short-acting .beta..sub.2 agonists include
tachycardia, skeletal muscle tremor, hypokalemia, increased lactic
acid, headache, and hyperglycemia.
[0139] The CpG immunostimulatory oligonucleotides may be directly
administered to the subject or may be administered in conjunction
with a nucleic acid delivery complex. A nucleic acid delivery
complex shall mean a nucleic acid molecule associated with (e.g.
ionically or covalently bound to; or encapsulated within) a
targeting means (e.g. a molecule that results in higher affinity
binding to target cell. Examples of nucleic acid delivery complexes
include oligonucleotides associated with a sterol (e.g.
cholesterol), a lipid (e.g. a cationic lipid, virosome or
liposome), or a target cell specific binding agent (e.g. a ligand
recognized by target cell specific receptor). Preferred complexes
may be sufficiently stable in vivo to prevent significant
uncoupling prior to internalization by the target cell. However,
the complex can be cleavable under appropriate conditions within
the cell so that the nucleic acid is released in a functional
form.
[0140] Delivery vehicles or delivery devices for delivering antigen
and oligonucleotides to surfaces have been described. The CpG
immunostimulatory oligonucleotide and/or the antigen and/or other
therapeutics may be administered alone (e.g., in saline or buffer)
or using any delivery vehicles known in the art. For instance the
following delivery vehicles have been described: Cochleates;
Emulsomes, ISCOMs; Liposomes; Live bacterial vectors (e.g.,
Salmonella, Escherichia coli, Bacillus calmatte-guerin, Shigella,
Lactobacillus); Live viral vectors (e.g., Vaccinia, adenovirus,
Herpes Simplex); Microspheres; Oligonucleotide vaccines; Polymers;
Polymer rings; Proteosomes; Sodium Fluoride; Transgenic plants;
Virosomes; Virus-like particles. Other delivery vehicles are known
in the art and some additional examples are provided below in the
discussion of vectors.
[0141] The term effective amount of a CpG immunostimulatory
oligonucleotide refers to the amount necessary or sufficient to
realize a desired biologic effect. For example, an effective amount
is that amount sufficient to reduce or prevent further induction of
viral load in order to avoid exacerbation of asthma. Combined with
the teachings provided herein, by choosing among the various active
compounds and weighing factors such as potency, relative
bioavailability, patient body weight, severity of adverse
side-effects and preferred mode of administration, an effective
prophylactic or therapeutic treatment regimen can be planned which
does not cause substantial toxicity and yet is entirely effective
to treat the particular subject. The effective amount for any
particular application can vary depending on such factors as the
disease or condition being treated, the particular CpG
immunostimulatory oligonucleotide being administered the size of
the subject, or the severity of the disease or condition. One of
ordinary skill in the art can empirically determine the effective
amount of a particular CpG immunostimulatory oligonucleotide and/or
other therapeutic agent without necessitating undue
experimentation.
[0142] Subject doses of the compounds described herein for mucosal
or local delivery typically range from about 0.1 .mu.g to 10 mg per
administration, which depending on the application could be given
daily, weekly, or monthly and any other amount of time
therebetween. More typically mucosal or local doses range from
about 10 .mu.g to 5 mg per administration, and most typically from
about 100 .mu.g to 1 mg, with 2-4 administrations being spaced days
or weeks apart. More typically, immune stimulant doses range from 1
.mu.g to 10 mg per administration, and most typically 10 .mu.g to 1
mg, with daily or weekly administrations. Subject doses of the
compounds described herein for parenteral delivery for the purpose
of inducing an immune response may be typically 5 to 10,000 times
higher than the effective mucosal dose, and more typically 10 to
1,000 times higher, and most typically 20 to 100 times higher.
Doses of the compounds described herein for parenteral delivery for
the purpose of inducing an innate immune response or for inducing
an immune response when the CpG immunostimulatory oligonucleotides
are administered in combination with other therapeutic agents or in
specialized delivery vehicles typically range from about 0.1 .mu.g
to 10 mg per administration, which depending on the application
could be given daily, weekly, or monthly and any other amount of
time therebetween. More typically parenteral doses for these
purposes range from about 10 .mu.g to 5 mg per administration, and
most typically from about 100 .mu.g to 1 mg, with 2-4
administrations being spaced days or weeks apart. In some
embodiments, however, parenteral doses for these purposes may be
used in a range of 5 to 10,000 times higher than the typical doses
described above. The oligonucleotides may be administered in
multiple doses over extended period of time.
[0143] For any compound described herein the therapeutically
effective amount can be initially determined from animal models. A
therapeutically effective dose can also be determined from human
data for CpG oligonucleotides which have been tested in humans
(human clinical trials have been initiated) and for compounds which
are known to exhibit similar pharmacological activities, such as
other adjuvants, e.g., LT and other antigens for vaccination
purposes. Higher doses may be required for parenteral
administration. The applied dose can be adjusted based on the
relative bioavailability and potency of the administered compound.
Adjusting the dose to achieve maximal efficacy based on the methods
described above and other methods as are well-known in the art is
well within the capabilities of the ordinarily skilled artisan.
[0144] The formulations of the invention are administered in
pharmaceutically acceptable solutions, which may routinely contain
pharmaceutically acceptable concentrations of salt, buffering
agents, preservatives, compatible carriers, adjuvants, and
optionally other therapeutic ingredients.
[0145] For use in therapy, an effective amount of the CpG
immunostimulatory oligonucleotide can be administered to a subject
by any mode that delivers the oligonucleotide to the desired
surface, e.g., mucosal, systemic. Administering the pharmaceutical
composition of the present invention may be accomplished by any
means known to the skilled artisan. Preferred routes of
administration include but are not limited to oral, parenteral,
intramuscular, intranasal, sublingual, intratracheal, inhalation,
ocular, vaginal, and rectal.
[0146] For oral administration, the compounds (i.e., CpG
immunostimulatory oligonucleotides and other therapeutic agents)
can be formulated readily by combining the active compound(s) with
pharmaceutically acceptable carriers well known in the art. Such
carriers enable the compounds of the invention to be formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions and the like, for oral ingestion by a subject to be
treated. Pharmaceutical preparations for oral use can be obtained
as solid excipient, optionally grinding a resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers such as sugars,
including lactose, sucrose, mannitol, or sorbitol; cellulose
preparations such as, for example, maize starch, wheat starch, rice
starch, potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate. Optionally the oral formulations may also be formulated
in saline or buffers, i.e. EDTA for neutralizing internal acid
conditions or may be administered without any carriers.
[0147] Also specifically contemplated are oral dosage forms of the
above component or components. The component or components may be
chemically modified so that oral delivery of the derivative is
efficacious. Generally, the chemical modification contemplated is
the attachment of at least one moiety to the component molecule
itself, where said moiety permits (a) inhibition of proteolysis;
and (b) uptake into the blood stream from the stomach or intestine.
Also desired is the increase in overall stability of the component
or components and increase in circulation time in the body.
Examples of such moieties include: polyethylene glycol, copolymers
of ethylene glycol and propylene glycol, carboxymethyl cellulose,
dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline.
Abuchowski and Davis, 1981, "Soluble Polymer-Enzyme Adducts" In:
Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience,
New York, N.Y., pp. 367-383; Newmark, et al., 1982, J. Appl.
Biochem. 4:185-189. Other polymers that could be used are
poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for
pharmaceutical usage, as indicated above, are polyethylene glycol
moieties.
[0148] For the component (or derivative) the location of release
may be the stomach, the small intestine (the duodenum, the jejunum,
or the ileum), or the large intestine. One skilled in the art has
available formulations which will not dissolve in the stomach, yet
will release the material in the duodenum or elsewhere in the
intestine. Preferably, the release will avoid the deleterious
effects of the stomach environment, either by protection of the
oligonucleotide (or derivative) or by release of the biologically
active material beyond the stomach environment, such as in the
intestine.
[0149] To ensure full gastric resistance a coating impermeable to
at least pH 5.0 is essential. Examples of the more common inert
ingredients that are used as enteric coatings are cellulose acetate
trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP),
HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit
L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L,
Eudragit S, and Shellac. These coatings may be used as mixed
films.
[0150] A coating or mixture of coatings can also be used on
tablets, which are not intended for protection against the stomach.
This can include sugar coatings, or coatings which make the tablet
easier to swallow. Capsules may consist of a hard shell (such as
gelatin) for delivery of dry therapeutic i.e. powder; for liquid
forms, a soft gelatin shell may be used. The shell material of
cachets could be thick starch or other edible paper. For pills,
lozenges, molded tablets or tablet triturates, moist massing
techniques can be used.
[0151] The therapeutic can be included in the formulation as fine
multi-particulates in the form of granules or pellets of particle
size about 1 mm. The formulation of the material for capsule
administration could also be as a powder, lightly compressed plugs
or even as tablets. The therapeutic could be prepared by
compression.
[0152] Colorants and flavoring agents may all be included. For
example, the oligonucleotide (or derivative) may be formulated
(such as by liposome or microsphere encapsulation) and then further
contained within an edible product, such as a refrigerated beverage
containing colorants and flavoring agents.
[0153] One may dilute or increase the volume of the therapeutic
with an inert material. These diluents could include carbohydrates,
especially mannitol, a-lactose, anhydrous lactose, cellulose,
sucrose, modified dextrans and starch. Certain inorganic salts may
be also be used as fillers including calcium triphosphate,
magnesium carbonate and sodium chloride. Some commercially
available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and
Avicell.
[0154] Disintegrants may be included in the formulation of the
therapeutic into a solid dosage form. Materials used as
disintegrates include but are not limited to starch, including the
commercial disintegrant based on starch, Explotab. Sodium starch
glycolate, Amberlite, sodium carboxymethylcellulose,
ultramylopectin, sodium alginate, gelatin, orange peel, acid
carboxymethyl cellulose, natural sponge and bentonite may all be
used. Another form of the disintegrants are the insoluble cationic
exchange resins. Powdered gums may be used as disintegrants and as
binders and these can include powdered gums such as agar, Karaya or
tragacanth. Alginic acid and its sodium salt are also useful as
disintegrants.
[0155] Binders may be used to hold the therapeutic agent together
to form a hard tablet and include materials from natural products
such as acacia, tragacanth, starch and gelatin. Others include
methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl
cellulose (CMC). Polyvinyl pyrrolidone (PVP) and
hydroxypropylmethyl cellulose (HPMC) could both be used in
alcoholic solutions to granulate the therapeutic.
[0156] An anti-frictional agent may be included in the formulation
of the therapeutic to prevent sticking during the formulation
process. Lubricants may be used as a layer between the therapeutic
and the die wall, and these can include but are not limited to;
stearic acid including its magnesium and calcium salts,
polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and
waxes. Soluble lubricants may also be used such as sodium lauryl
sulfate, magnesium lauryl sulfate, polyethylene glycol of various
molecular weights, Carbowax 4000 and 6000.
[0157] Glidants that might improve the flow properties of the drug
during formulation and to aid rearrangement during compression
might be added. The glidants may include starch, talc, pyrogenic
silica and hydrated silicoaluminate.
[0158] To aid dissolution of the therapeutic into the aqueous
environment a surfactant might be added as a wetting agent.
Surfactants may include anionic detergents such as sodium lauryl
sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium
sulfonate. Cationic detergents might be used and could include
benzalkonium chloride or benzethomium chloride. The list of
potential non-ionic detergents that could be included in the
formulation as surfactants are lauromacrogol 400, polyoxyl 40
stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60,
glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty
acid ester, methyl cellulose and carboxymethyl cellulose. These
surfactants could be present in the formulation of the
oligonucleotide or derivative either alone or as a mixture in
different ratios.
[0159] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. Microspheres formulated for oral
administration may also be used. Such microspheres have been well
defined in the art. All formulations for oral administration should
be in dosages suitable for such administration.
[0160] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0161] For administration by inhalation, the compounds for use
according to the present invention may be conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0162] Also contemplated herein is pulmonary delivery of the
oligonucleotides (or derivatives thereof). The oligonucleotide (or
derivative) is delivered to the lungs of a mammal while inhaling
and traverses across the lung epithelial lining to the blood
stream. Other reports of inhaled molecules include Adjei et al.,
1990, Pharmaceutical Research, 7:565-569; Adjei et al., 1990,
International Journal of Pharmaceutics, 63:135-144 (leuprolide
acetate); Braquet et al., 1989, Journal of Cardiovascular
Pharmacology, 13(suppl. 5):143-146 (endothelin-1); Hubbard et al.,
1989, Annals of Internal Medicine, Vol. III, pp. 206-212
(a1-antitrypsin); Smith et al., 1989, J. Clin. Invest. 84:1145-1146
(a-1-proteinase); Oswein et al., 1990, "Aerosolization of
Proteins", Proceedings of Symposium on Respiratory Drug Delivery
II, Keystone, Colo., March, (recombinant human growth hormone);
Debs et al., 1988, J. Immunol. 140:3482-3488 (interferon-g and
tumor necrosis factor alpha) and Platz et al., U.S. Pat. No.
5,284,656 (granulocyte colony stimulating factor). A method and
composition for pulmonary delivery of drugs for systemic effect is
described in U.S. Pat. No. 5,451,569, issued Sep. 19, 1995 to Wong
et al.
[0163] Contemplated for use in the practice of this invention are a
wide range of mechanical devices designed for pulmonary delivery of
therapeutic products, including but not limited to nebulizers,
metered dose inhalers, and powder inhalers, all of which are
familiar to those skilled in the art.
[0164] Some specific examples of commercially available devices
suitable for the practice of this invention are the Ultravent
nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the
Acorn II nebulizer, manufactured by Marquest Medical Products,
Englewood, Colo.; the Ventolin metered dose inhaler, manufactured
by Glaxo Inc., Research Triangle Park, N.C.; and the Spinhaler
powder inhaler, manufactured by Fisons Corp., Bedford, Mass.
[0165] All such devices require the use of formulations suitable
for the dispensing of oligonucleotide (or derivative). Typically,
each formulation is specific to the type of device employed and may
involve the use of an appropriate propellant material, in addition
to the usual diluents, adjuvants and/or carriers useful in therapy.
Also, the use of liposomes, microcapsules or microspheres,
inclusion complexes, or other types of carriers is contemplated.
Chemically modified oligonucleotide may also be prepared in
different formulations depending on the type of chemical
modification or the type of device employed.
[0166] Formulations suitable for use with a nebulizer, either jet
or ultrasonic, will typically comprise oligonucleotide (or
derivative) dissolved in water at a concentration of about 0.1 to
25 mg of biologically active oligonucleotide per mL of solution.
The formulation may also include a buffer and a simple sugar (e.g.,
for oligonucleotide stabilization and regulation of osmotic
pressure). The nebulizer formulation may also contain a surfactant,
to reduce or prevent surface induced aggregation of the
oligonucleotide caused by atomization of the solution in forming
the aerosol.
[0167] Formulations for use with a metered-dose inhaler device will
generally comprise a finely divided powder containing the
oligonucleotide (or derivative) suspended in a propellant with the
aid of a surfactant. The propellant may be any conventional
material employed for this purpose, such as a chlorofluorocarbon, a
hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,
including trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or
combinations thereof. Suitable surfactants include sorbitan
trioleate and soya lecithin. Oleic acid may also be useful as a
surfactant.
[0168] Formulations for dispensing from a powder inhaler device
will comprise a finely divided dry powder containing
oligonucleotide (or derivative) and may also include a bulking
agent, such as lactose, sorbitol, sucrose, or mannitol in amounts
which facilitate dispersal of the powder from the device, e.g., 50
to 90% by weight of the formulation. The oligonucleotide (or
derivative) should most advantageously be prepared in particulate
form with an average particle size of less than 10 mm (or microns),
most preferably 0.5 to 5 mm, for most effective delivery to the
distal lung.
[0169] Nasal delivery of a pharmaceutical composition of the
present invention is also contemplated. Nasal delivery allows the
passage of a pharmaceutical composition of the present invention to
the blood stream directly after administering the therapeutic
product to the nose, without the necessity for deposition of the
product in the lung. Formulations for nasal delivery include those
with dextran or cyclodextran.
[0170] For nasal administration, a useful device is a small, hard
bottle to which a metered dose sprayer is attached. In one
embodiment, the metered dose is delivered by drawing the
pharmaceutical composition of the present invention solution into a
chamber of defined volume, which chamber has an aperture
dimensioned to aerosolize and aerosol formulation by forming a
spray when a liquid in the chamber is compressed. The chamber is
compressed to administer the pharmaceutical composition of the
present invention. In a specific embodiment, the chamber is a
piston arrangement. Such devices are commercially available.
[0171] Alternatively, a plastic squeeze bottle with an aperture or
opening dimensioned to aerosolize an aerosol formulation by forming
a spray when squeezed is used. The opening is usually found in the
top of the bottle, and the top is generally tapered to partially
fit in the nasal passages for efficient administration of the
aerosol formulation. Preferably, the nasal inhaler will provide a
metered amount of the aerosol formulation, for administration of a
measured dose of the drug.
[0172] The compounds, when it is desirable to deliver them
systemically, may be formulated for parenteral administration by
injection, e.g., by bolus injection or continuous infusion.
Formulations for injection may be presented in unit dosage form,
e.g., in ampoules or in multi-dose containers, with an added
preservative. The compositions may take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and may contain
formulatory agents such as suspending, stabilizing and/or
dispersing agents.
[0173] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0174] Alternatively, the active compounds may be in powder form
for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0175] The compounds may also be formulated in rectal or vaginal
compositions such as suppositories or retention enemas, e.g.,
containing conventional suppository bases such as cocoa butter or
other glycerides.
[0176] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt.
[0177] The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include but are not limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene
glycols.
[0178] Suitable liquid or solid pharmaceutical preparation forms
are, for example, aqueous or saline solutions for inhalation,
microencapsulated, encochleated, coated onto microscopic gold
particles, contained in liposomes, nebulized, aerosols, pellets for
implantation into the skin, or dried onto a sharp object to be
scratched into the skin. The pharmaceutical compositions also
include granules, powders, tablets, coated tablets,
(micro)capsules, suppositories, syrups, emulsions, suspensions,
creams, drops or preparations with protracted release of active
compounds, in whose preparation excipients and additives and/or
auxiliaries such as disintegrants, binders, coating agents,
swelling agents, lubricants, flavorings, sweeteners or solubilizers
are customarily used as described above. The pharmaceutical
compositions are suitable for use in a variety of drug delivery
systems. For a brief review of methods for drug delivery, see
Langer, Science 249:1527-1533, 1990, which is incorporated herein
by reference.
[0179] The CpG immunostimulatory oligonucleotides and optionally
other therapeutics may be administered per se (neat) or in the form
of a pharmaceutically acceptable salt. When used in medicine the
salts should be pharmaceutically acceptable, but
non-pharmaceutically acceptable salts may conveniently be used to
prepare pharmaceutically acceptable salts thereof. Such salts
include, but are not limited to, those prepared from the following
acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric,
maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric,
methane sulphonic, formic, malonic, succinic,
naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts
can be prepared as alkaline metal or alkaline earth salts, such as
sodium, potassium or calcium salts of the carboxylic acid
group.
[0180] Suitable buffering agents include: acetic acid and a salt
(1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a
salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).
Suitable preservatives include benzalkonium chloride (0.003-0.03%
w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and
thimerosal (0.004-0.02% w/v).
[0181] The term pharmaceutically-acceptable carrier means one or
more compatible solid or liquid filler, diluents or encapsulating
substances which are suitable for administration to a human or
other vertebrate animal. The term carrier denotes an organic or
inorganic ingredient, natural or synthetic, with which the active
ingredient is combined to facilitate the application. The
components of the pharmaceutical compositions also are capable of
being commingled with the compounds of the present invention, and
with each other, in a manner such that there is no interaction
which would substantially impair the desired pharmaceutical
efficiency.
[0182] The present invention is further illustrated by the
following Examples, which in no way should be construed as further
limiting. The entire contents of all of the references (including
literature references, issued patents, published patent
applications, and co-pending patent applications) cited throughout
this application are hereby expressly incorporated by
reference.
EXAMPLES
Example 1
[0183] 1. Induction of IFN.alpha. and IFN-Associated Genes by a
C-class CpG ODN (SEQ ID NO:10)
[0184] Methods: Mice (male, BALB/c) received SEQ ID NO:10 (100
.mu.g/kg in FIGS. 3a-3c or 10, 100, or 1000 .mu.l/kg in FIGS.
3d-3f) or saline by intranasal instillation. Secreted proteins (IFN
.alpha., IFN .gamma., and IP10) were assayed in bronchoalveolar
lavage fluid 15 hours later, or gene expression in lung tissue was
analyzed by real-time PCR 30 hours later.
[0185] Results: C-class CpG ODN induced secretion of IFN.alpha.,
IFN.gamma. and interferon-inducible protein-10 (IP-10). The results
are shown in FIG. 3.
[0186] Since the CpG ODN stimulated secretion of IFN.alpha. in
mouse airways, we investigated whether the interferon-inducible
gene for indoleamine 2,3 dioxygenase becomes expressed in the lung.
When instilled in the airways, the CpG ODN did increase expression
of mRNA for this immune-modulating enzyme (FIG. 3f). Mx1, and
indoleamine 2,3-dioxygenase were also upregulated in mouse lung
(FIGS. 3d and 3e).
[0187] 2. Anti-Viral Effects of a C-class CpG ODN
[0188] Because respiratory tract virus infections are a major cause
of asthma exacerbations, a mouse model was established in which
airway inflammation is exacerbated by combined antigen challenge
and virus infection.
[0189] Methods Mice received two adminsitrations of SEQ ID NO:10 at
30, 100, or 300 .mu.g/kg, 4 days apart, by intranasal instillation
in 40 .mu.l saline. Two days after the last dose, mice were
infected with influenza virus (influenza type A, subtype H1N1,
mouse adapted strain PR8, 200 EID50, in 40 .mu.l saline) by
intranasal instillation.
[0190] Virus load in the lung (Takara Biomedical enzyme immunoassay
for nuclear protein) and airway inflammation (counts of cells
recovered by bronchoalveolar lavage) were assessed 6 days after
virus infection.
[0191] Results: Pretreatment with SEQ ID NO: 10 reduced influenza
virus load in the lung (FIG. 4a) and virus-induced accumulation of
leukocytes (including neutrophils and mononuclear cells) in the
airways (FIGS. 4b and 4c). The results are shown in FIG. 4.
[0192] 3. Protective Effects of a C-Class CpG ODN Against Antigen-
and Virus-Induced Airway Inflammation and Hyperreactivty
[0193] Methods Mice were sensitized with antigen (cockroach, 10
.mu.g, intraperitoneal with aluminum hydroxide adjuvant) and then
challenged twice each week for three weeks with intranasal antigen
(10 .mu.g in 40 .mu.l saline). Mice were infected with influenza
virus by intranasal instillation before the last pair of antigen
challenges. Alternatively, separate mice received antigen challenge
alone or virus infection alone.
[0194] SEQ ID NO:10 (100 .mu.g/kg) was administered intranasally
once each week, two days before the first antigen challenge of the
week. Airway inflammation (counts of cells recovered by
bronchoalveolar lavage) and airway hyperreactivity to inhaled
methacholine (Sigma, St. Louis, Mo., USA) were assessed 48 hours
after the last antigen challenge. Mice were anaesthetized with
sodium pentobarbitone (60 mg/kg, intraperitoneal) and mechanically
ventilated through a tracheal cannula. Cells were recovered from
the airways by bronchoalveolar lavage performed with 1 ml of RPMI
1640 medium containing 10% fetal bovine serum (both from
Invitrogen, Carlsbad, Calif., USA) instilled through a tracheal
cannula. Airway resistance was calculated from measurements of
pulmonary airflow and intratracheal pressure using respiratory
mechanics software (Buxco Research Systems, Wilmington, N.C., USA).
After recording baseline airway resistance, increasing
concentrations of methacholine aerosol (5-100 mg/ml for 5 seconds,
at 5 minute intervals) were delivered through the tracheal cannula.
The resulting bronchoconstriction was measured as increase in
airway resistance. For each animal, the area under the methacholine
dose-response curve was calculated.
[0195] Analysis of data: Statistical significance of differences
between treatment group and untreated control group means were
determined using the Mann-Whitney test or Kruskal-Wallis multiple
comparison test (* P<0.05).
[0196] Results: Mice that were both antigen-challenged and
virus-infected showed a more severe accumulation of leukocytes
(including neutrophils and mononuclear cells) in the airways than
mice that were either antigen challenged alone or virus infected
alone (FIGS. 5a-5c).
[0197] These mice also developed airway hyperreactivity. When dosed
into the airways once each week for three weeks, the CpG ODN
protected mice against the exacerbated airways inflammation and the
fall in body weight, and almost completely prevented the increase
in baseline airway resistance and the development of airway
hyperreactivity (FIGS. 6a-6c).
Example 2
[0198] It has been demonstrated that the class C CpG
oligodeoxynucleotide can suppress influenza virus load and
virus-induced airway inflammation in mice. In Example 2 the
protective effects of SEQ ID NO: 10 against the exacerbated airway
inflammation induced by combined influenza virus infection and
antigen challenge were examined.
[0199] Methods
[0200] 1. Antigen and Virus Administrations:
[0201] Mice (male BALB/c) were sensitized on study days 0 and 7
with antigen (cockroach, 10 .mu.g, intraperitoneal) with aluminum
hydroxide adjuvant (Pierce Alum).
[0202] Mice were antigen challenged by exposure to
intranasally-administered antigen (10 .mu.g in 40 .mu.l saline),
twice each week for three consecutive weeks. The first challenge
was on study day 21.
[0203] Mice were infected with influenza virus (influenza type A,
subtype H1N1, mouse adapted strain PR8, 200 EID.sub.50 in 40 .mu.l
saline) by intranasal instillation on study day 34 (i.e. before the
last pair of antigen challenges).
[0204] Alternatively, separate groups of mice received antigen
challenge alone or virus infection alone.
[0205] 2. Treatment with SEQ ID NO:10:
[0206] SEQ ID NO:10 (100 .mu.g/kg) was administered intranasally
once each week, two days before the first antigen challenge of the
week.
[0207] 3. Endpoints:
[0208] Airway inflammation was assessed 48 hours after the last
antigen challenge. Cells in airways were recovered by
bronchoalveolar lavage. Differential cell counts were made by light
microscopy from cytocentrifuge preparations stained with
Wright-Giemza stain.
[0209] Summary of Study Protocol TABLE-US-00002 TABLE 2 Virus
.dwnarw. Antigen Antigen Antigen Antigen sensitize challenge
challenge challenge .dwnarw. .dwnarw. .dwnarw. .dwnarw. .dwnarw.
.dwnarw. .dwnarw. .dwnarw. ODN ODN ODN .dwnarw. .dwnarw. .dwnarw.
Day: 0 7 19 21 24 26 28 31 33 34 35 38 40 .dwnarw. Endpoints First
Second Third treatment week treatment week treatment week
[0210] Results
[0211] Characterization of Virus- and Antigen-Induced Airway
Inflammation
[0212] Infection with influenza virus alone or antigen challenge
alone each caused an increase in the total number of leukocytes in
bronchoalveolar lavage fluid (FIG. 7). In virus-infected mice, this
cell accumulation included a marked neutrophilia, whereas in
antigen-challenged mice, the accumulation included a marked
eosinophilia.
[0213] When compared with mice that received antigen challenge
alone, those that were antigen-challenged and virus-infected showed
an exacerbated accumulation of leukocytes in bronchoalveolar lavage
fluid (FIG. 7). This increased accumulation included both
neutrophils and mononuclear cells. However, these mice showed
reduced eosinophilia.
[0214] Effects of SEQ ID NO: 10:
[0215] Treatment with SEQ ID NO:10 (100 .mu.g/kg) did not suppress
the virus-induced neutrophilia (FIG. 7). This finding was expected
at this dose. It has been determined that a higher dose of 300
.mu.g/kg generally demonstrates better anti-virus effects.
[0216] In contrast, SEQ ID NO: 10 (100 .mu.g/kg) significantly
suppressed antigen-induced cellular infiltration (FIG. 7).
[0217] An important finding of this study was that SEQ ID NO:10
(100 .mu.g/kg) significantly suppressed the exacerbated airway
inflammation induced in mice that were both virus-infected and
antigen-challenged. The exacerbated accumulations of neutrophils
and mononuclear cells were both suppressed (FIG. 7).
[0218] In addition to exacerbated airway inflammation, mice that
were both virus-infected and antigen-challenged showed a marked
loss of body weight. This was significantly suppressed in mice
treated with SEQ ID NO: 10.
Example 3
[0219] Induction of TLR9-Associated Cytokines from Mouse
Splenocytes In Vitro, and in the Mouse Lung In Vivo
[0220] The ability of SEQ ID NO: 10 to induce secretion of
TLR9-associated cytokines from murine splenocytes in vitro was
examined.
[0221] Methods
[0222] Stimulation of Cytokines from Splenocytes In Vitro
[0223] Splenocytes were pooled from 6 mice and incubated with ODNs
(0.1, 1 or 10 .mu.g/ml) for 36 hours. Cells were isolated
mechanically by gently pushing chopped mouse spleens through a cell
sieve (70 .mu.m pore size). Cells (1.times.10.sup.7, pooled from 6
mice) were incubated (37.degree. C., 5% CO.sub.2) in 1 ml medium
(RPMI 1640 containing 10% fetal bovine serum, both from Invitrogen,
Carlsbad, Calif., USA). SEQ ID NO: 10 or control ODN (with reversed
CpG motifs) or either of the two domains of SEQ ID NO: 10 (5' end
stimulatory sequence and palindrome) were added to give
concentrations of 0.1, 1 or 10 .mu.g/ml. After incubation for 24
hours, culture medium was assayed as described below for secreted
cytokines (IFN.alpha., IFN.gamma., interferon-inducible protein
[IP]-10, IL-6, IL-10 and TNF.alpha.).
[0224] Stimulation of Cytokines in Mouse Airways
[0225] Mice received SEQ ID NO:10 (10-1000 .mu.g/kg) or vehicle (40
.mu.l saline) delivered into the airways by intranasal instillation
carried out under light isoflurane anaesthesia. Twenty-four hours
later, bronchoalveolar lavage was performed through a tracheal
cannula using 1 ml of saline. Cytokine concentrations (IFN.alpha.,
IFN.gamma., IP-10, IL-6 and IL-12p40) in bronchoalveloar lavage
fluid were assayed.
[0226] Results
[0227] As shown in table one, SEQ ID NO: 10 induced secretion of
TLR9-associated cytokines from isolated murine splenocytes. In
contrast, a control ODN with reversed CpG motifs, the 5' end
stimulatory sequence of SEQ ID NO: 10 alone, or the palindrome
alone had no marked activity. The highest titers of each cytokine
were induced with ODNs at 10 .mu.g/ml (data from lower
concentrations are not shown). n.d.=not detected (<12 pg/ml).
Thus, SEQ ID NO: 10 induced secretions of IFN.alpha., IFN.gamma.,
IP-10, IL-6, IL-10 and TNF.alpha. in a concentration-dependent
manner. The highest titers of each cytokine were induced with SEQ
ID NO: 10 at 10 .mu.g/ml.
[0228] To evaluate the importance of correctly-ordered CpG motifs
for this biological activity of SEQ ID NO:10, the assay was
repeated with an ODN with the same sequence as SEQ ID NO:10 but
with reversed CpG motifs in the 5' end stimulatory sequence (SEQ ID
NO: 55). The control oligonucleotide showed almost no ability to
induce these TLR9-associated cytokines. The 5' end stimulatory
sequence of SEQ ID NO:10 alone, or the palindrome alone, also had
no marked activity demonstrating that an intact molecule with both
these domains is required for activity (sequences shown in Table
3). TABLE-US-00003 TABLE 3 Cytokine titer (pg/ml) induced by ODN
(10 .mu.g/ml) ODN IFN.alpha. IFN.gamma. IP-10 IL-6 IL-10 TNF.alpha.
Medium alone 34.4 n.d. 21.9 64.8 n.d. n.d. SEQ ID NO: 10 199.0
539.9 111.1 11531.5 113.1 71.1 Control ODN 22.1 n.d. 22.4 537.5
n.d. n.d. Stimulatory 19.5 n.d. 17.9 427.1 n.d. 21.0 sequence alone
Palindrome alone 18.8 n.d. 17.7 59.0 n.d. n.d.
[0229] It was then investigated whether SEQ ID NO:10 could induce
TLR9-associated cytokines when dosed into mouse airways in vivo.
SEQ ID NO: 10 induced secretion of IFN.alpha.c, IFN.gamma., IP-10,
IL-6 and IL-12p40 as demonstrated by increased concentrations of
these cytokines in bronchoalveolar lavage fluid (FIG. 8).
Example 4
[0230] SEQ ID NO: 10 Induces Immune Deviation Away from a Th2
Response to Antigen Sensitization.
[0231] To determine whether SEQ ID NO: 10 could suppress a Th2
response to antigen sensitization when injected into the mouse
footpad together with a sensitizing antigen (ovalbumin), the mice
were re-stimulated with antigen in a recall assay ex vivo.
[0232] Methods
[0233] Mice were sensitized with antigen (10 .mu.g grade V
ovalbumin, Sigma, St. Louis, Mo., USA) injected into the right rear
footpad. Antigen was injected either alone, or together with SEQ ID
NO:10 (10-1000 .mu.g/kg). In each case, total injection volume was
20 .mu.l. Six days later, the draining popliteal lymph node was
removed and a cell suspension was prepared by gently pushing the
nodes through a cell sieve (70 .mu.m pore size). An antigen recall
assay was carried out ex vivo by incubating (37.degree. C., 5%
CO.sub.2) 1.times.10.sup.6 unfractionated lymph node cells in 220
.mu.l medium (RPMI 1640 containing 10% fetal bovine serum, both
from Invitrogen, Carlsbad, Calif., USA) in the presence or absence
of antigen (ovalbumin, 10 .mu.g/ml). After incubation for 36 hours,
culture medium was assayed as described below for secreted
cytokines (IL-5, IL-13 and IFN.gamma.).
[0234] Results
[0235] Popliteal lymph node cells from sensitized mice secreted
IL-5, IL-13 and IFN.gamma. (FIG. 9). Cells incubated in the absence
of antigen, or with a control antigen (cockroach) to which the mice
had not been sensitized, did not secrete detectable titers of any
of these cytokines (<19 pg/ml). Cells isolated from SEQ ID NO:
10-treated animals showed attenuated antigen-induced secretions of
the Th2 cytokines IL-5 and IL-13. In contrast, secretion of the Th1
cytokine IFN.gamma. was markedly increased (FIG. 9c). Cells
incubated in the absence of antigen, or with a control antigen
(cockroach) to which the mice had not been sensitized, did not
secrete detectable titers of any of these cytokines (<10
pg/ml).
Example 5
[0236] SEQ ID NO: 10 Suppresses Antigen-Induced IgE Production and
Stimulates IgG2a Production in the Mouse In Vivo.
[0237] It was next determined whether SEQ ID NO: 10 could alter the
profile of immunoglobulin production when dosed to mice at the time
of antigen sensitization.
[0238] Methods
[0239] Mice were antigen sensitized twice, 7 days apart, with
intraperitoneal antigen (10 .mu.g grade V ovalbumin, Sigma, St.
Louis, Mo., USA) dissolved in aluminum hydroxide adjuvant (0.2 ml,
Pierce Imject Alum, Rockford, Ill., USA). Mice received SEQ ID
NO:10 (1-1000 .mu.g/kg) or control vehicle (saline, 10 ml/kg) by
intraperitoneal injection two days before each of the two
sensitizations, and on the day of each sensitization. Mice were
bled by cardiac puncture 12 days after the second sensitization.
Serum was collected by centrifugation and assayed as follows for
ovalbumin-specific IgE and IgG2a.
[0240] ELISAs were carried out in microtiter plates (Nunc,
Rochester, N.Y., USA), with washes using 0.05% polysorbate 20
(Sigma, St. Louis, Mo., USA) in phosphate buffered saline
(Invitrogen, Carlsbad, Calif., USA) between each of the following
steps. Plates were coated with ovalbumin (150 .mu.l of 100
.mu.g/ml) in binding buffer (0.1M NaHCO.sub.3 Sigma) for 15 hours
at 4.degree. C. Plates were then blocked with assay diluent (200
.mu.l/well, Pharmingen, BD Biosciences, Franklin Lakes, N.J., USA)
for 2 hours at 20.degree. C. Serum samples (diluted 1 in 40 in
assay diluent, 100 .mu.l/well) were added and left for 2 hours at
20.degree. C. Biotin-conjugated rat anti-mouse IgE or IgG2a
(Pharmingen) (2 .mu.g/ml in assay diluent, 100 .mu.l/well) were
added and left for 2 hours at 4.degree. C. Streptavidin-conjugated
horseradish peroxidase (Pharmingen, diluted 1:1000 in assay
diluent, 100 .mu.l/well) was then added and left for 1 hour at
20.degree. C. Tetramethyl benzidine substrate reagent (Pharmingen,
100 .mu.l/well) was added for 30 minutes at 20.degree. C. and the
reaction was then stopped with 2N sulphuric acid (50 .mu.l/well).
Absorbance at 450 nm was measured using a spectrophotometer
(Spectramax, Molecular Devices, Sunnyvale, Calif., USA).
Results
[0241] SEQ ID NO:10 suppressed the production of antigen-specific
IgE (85% suppression with a dose of 1000 .mu.g/kg) and potentiated
the production of IgG2a providing further evidence of immune
deviation away from a Th2 response to antigen (FIG. 10).
Example 6
[0242] SEQ ID NO: 10 Suppresses Antigen-Induced Accumulations of
Eosinophils and Lymphocytes in Mouse Airways In Vivo.
[0243] Examples 4 and 5 demonstrate that SEQ ID NO: 10 is able to
suppress Th2 immune responses. Therefore, the protective effects of
SEQ ID NO: 10 in a mouse model of antigen-induced airway
inflammation were examined.
[0244] Methods
[0245] Mice were sensitized with intraperitoneal antigen
(cockroach) and then antigen challenged twice a week for 2 weeks
with antigen instilled into the airways. During each of the 2
challenge weeks, mice were treated once with SEQ ID NO:10 or
vehicle (Veh) instilled into the airways. Alternatively, mice were
untreated (Untr). Bronchoalveolar lavage was performed 48 hours
after the last antigen challenge and recovered cells were counted.
Total leukocytes (a) and eosinophils (b) were counted with an
automated cell counter.
[0246] Results
[0247] As this experimental model shares hallmark features of
allergic asthma, the protective effects of SEQ ID NO:10 for this
indication were examined When dosed into the airways once each week
for two weeks, SEQ ID NO:10 suppressed the airway accumulations of
eosinophils, T cells and B cells that were induced by
intrapulmonary antigen challenge (FIG. 11). At the highest dose
tested (300 .mu.g/kg), SEQ ID NO:10 suppressed accumulations of
these cells by 78%, 65% and 79% respectively.
CONCLUSIONS
[0248] In both children and adults with existing asthma, infections
with respiratory tract viruses are important precipitants for
airway obstruction and wheezing. The inflammatory processes
involved are complex. However, virus-induced neutrophil and
mononuclear cell recruitment and activation are implicated in
aggravating the airway obstruction that contributes to these asthma
exacerbations. The data presented herein demonstrate that CpG ODN,
particularly C-class ODN, markedly suppress the exacerbated
accumulations of neutrophils and mononuclear cells induced in mice
by combined virus infection and antigen challenge.
[0249] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
examples provided, since the examples are intended as a single
illustration of one aspect of the invention and other functionally
equivalent embodiments are within the scope of the invention.
Various modifications of the invention in addition to those shown
and described herein will become apparent to those skilled in the
art from the foregoing description and fall within the scope of the
appended claims. The advantages and objects of the invention are
not necessarily encompassed by each embodiment of the invention.
Sequence CWU 0
0
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 56 <210>
SEQ ID NO 1 <211> LENGTH: 22 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(2) <223> OTHER INFORMATION: wherein the
internucleotide linkage is a stablized internucleotide linkage
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (3)..(4) <223> OTHER INFORMATION: wherein the
internucleotide linkage is a stablized internucleotide linkage
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (5)..(7) <223> OTHER INFORMATION: wherein the
internucleotide linkages are stablized internucleotide linkages
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (8)..(11) <223> OTHER INFORMATION: wherein the
internucleotide linkages are stablized internucleotide linkages
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (12)..(16) <223> OTHER INFORMATION: wherein the
internucleotide linkages are stablized internucleotide linkages
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (17)..(22) <223> OTHER INFORMATION: wherein the
internucleotide linkages are stablized internucleotide linkages
<400> SEQUENCE: 1 tcgcgtcgtt cggcgcgcgc cg 22 <210> SEQ
ID NO 2 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(2)
<223> OTHER INFORMATION: wherein the internucleotide linkage
is a stabilized internucleotide linkage <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (3)..(5)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (6)..(8)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (9)..(12)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (13)..(17)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (18)..(23)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <400> SEQUENCE: 2
tcgtcgacgt tcggcgcgcg ccg 23 <210> SEQ ID NO 3 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(2) <223>
OTHER INFORMATION: wherein the internucleotide linkage is a
stabilized internucleotide linkage <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (3)..(6) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (11)..(15)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (16)..(21)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <400> SEQUENCE: 3
tcggacgttc ggcgcgcgcc g 21 <210> SEQ ID NO 4 <211>
LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(2) <223>
OTHER INFORMATION: wherein the internucleotide linkage is a
stabilized internucleotide linkage <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (3)..(6) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(10)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (11)..(19)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <400> SEQUENCE: 4
tcggacgttc ggcgcgccg 19 <210> SEQ ID NO 5 <211> LENGTH:
20 <212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(2) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a stabilized
internucleotide linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(4) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a stabilized
internucleotide linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (5)..(7) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a stabilized
internucleotide linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (8)..(11) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a stabilized
internucleotide linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (12)..(20) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a stabilized
internucleotide linkage <400> SEQUENCE: 5 tcgcgtcgtt
cggcgcgccg 20 <210> SEQ ID NO 6 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(2) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a stabilized
internucleotide linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(5) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (6)..(9) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (10)..(14) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (15)..(20) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <400> SEQUENCE: 6 tcgacgttcg
gcgcgcgccg 20 <210> SEQ ID NO 7 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(2) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a stabilized
internucleotide linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(5)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (6)..(9)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(18)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <400> SEQUENCE: 7
tcgacgttcg gcgcgccg 18 <210> SEQ ID NO 8 <211> LENGTH:
18 <212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(2) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a stabilized
internucleotide linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(4) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a stabilized
internucleotide linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (5)..(7) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (8)..(11) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (12)..(18) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <400> SEQUENCE: 8 tcgcgtcgtt
cggcgccg 18 <210> SEQ ID NO 9 <211> LENGTH: 22
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(2) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a stabilized
internucleotide linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(4) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a stabilized
internucleotide linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (5)..(7) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (8)..(11) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (12)..(16) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (17)..(22) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <400> SEQUENCE: 9 tcgcgacgtt
cggcgcgcgc cg 22 <210> SEQ ID NO 10 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(2) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a stabilized
internucleotide linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(5) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (6)..(8) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (9)..(12) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (13)..(17) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (18)..(23) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <400> SEQUENCE: 10 tcgtcgtcgt
tcggcgcgcg ccg 23 <210> SEQ ID NO 11 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(24) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <400> SEQUENCE: 11 tcgtcgtttt
gacgttttgt cgtt 24 <210> SEQ ID NO 12 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(24) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <400> SEQUENCE: 12 tcgtcgtttt
gtcgttttgt cgtt 24 <210> SEQ ID NO 13 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(5) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (6)..(12) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (13)..(18) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (19)..(24) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <400> SEQUENCE: 13 tcgtcgtttt
acggcgccgt gccg 24 <210> SEQ ID NO 14 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(2) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a stabilized
internucleotide linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(5) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (6)..(13) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(21) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (22)..(24) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <400> SEQUENCE: 14 tcgtcgtttt
gacgttttgt cgtt 24 <210> SEQ ID NO 15 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(2)
<223> OTHER INFORMATION: wherein the internucleotide linkage
is a stabilized internucleotide linkage <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (3)..(5)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (6)..(8)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (9)..(12)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (13)..(17)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (18)..(23)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <400> SEQUENCE: 15
tcgtcgacgt tcggcgcgcg ccg 23 <210> SEQ ID NO 16 <211>
LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(2) <223>
OTHER INFORMATION: wherein the internucleotide linkage is a
stabilized internucleotide linkage <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (12)..(15) <223>
OTHER INFORMATION: wherein the internucleotide linkage is a
stabilized internucleotide linkage <400> SEQUENCE: 16
tcgacgtcgt ggggg 15 <210> SEQ ID NO 17 <211> LENGTH: 19
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(19) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <400> SEQUENCE: 17 tcgacgtcga
cgtgacgtg 19 <210> SEQ ID NO 18 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(17) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <400> SEQUENCE: 18 tcgacgtcga
cgtgacg 17 <210> SEQ ID NO 19 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(3) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (4)..(6) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (7)..(11) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (12)..(14) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (15)..(17) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <400> SEQUENCE: 19 ttcgtcgttt
tgtcgtt 17 <210> SEQ ID NO 20 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(4) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (5)..(7) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (8)..(12) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (13)..(15) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (16)..(18) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <400> SEQUENCE: 20 tttcgtcgtt
tcgtcgtt 18 <210> SEQ ID NO 21 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(5) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (6)..(9) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (10)..(15) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (16)..(21) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <400> SEQUENCE: 21 tcgtcgtacg
gcgccgtgcc g 21 <210> SEQ ID NO 22 <211> LENGTH: 22
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(5) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (6)..(10) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (11)..(16) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (17)..(22) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <400> SEQUENCE: 22 tcgtcgttac
ggcgccgtgc cg 22 <210> SEQ ID NO 23 <211> LENGTH: 19
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(19) <223> OTHER
INFORMATION: wherein the internucleotide linkages are stabilized
internucleotide linkages <400> SEQUENCE: 23 tcgacgtcga
cgtgacgtt 19 <210> SEQ ID NO 24 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(5)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (6)..(8)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (9)..(12)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (13)..(18)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(24)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <400> SEQUENCE: 24
tcgtcgacga tcggcgccgt gccg 24 <210> SEQ ID NO 25 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(10) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (12)..(24)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <400> SEQUENCE: 25
tcgtcgacga tcggcgccgt gccg 24 <210> SEQ ID NO 26 <211>
LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(5) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (6)..(11)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (12)..(19)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <400> SEQUENCE: 26
tcgacgtcga cgtgacgtt 19 <210> SEQ ID NO 27 <211>
LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(5) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (6)..(13)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(19)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <400> SEQUENCE: 27
tcgacgtcga cgtgacgtt 19 <210> SEQ ID NO 28 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(5) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (6)..(11)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (12)..(17)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (18)..(24)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are stabilized internucleotide linkages <400> SEQUENCE: 28
tcgtcgttta cggcgccgtg ccgt 24 <210> SEQ ID NO 29 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(2) <223>
OTHER INFORMATION: where the internucleotide linkage is a
phosphorothioate internucleotide linkage <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (3)..(5)
<223> OTHER INFORMATION: where the internucleotide linkages
are phosphorothioate internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (6)..(13)
<223> OTHER INFORMATION: where the internucleotide linkages
are phosphorothioate internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(21)
<223> OTHER INFORMATION: where the internucleotide linkages
are phosphorothioate internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (22)..(24)
<223> OTHER INFORMATION: where the internucleotide linkages
are phosphorothioate internucleotide linkages <400> SEQUENCE:
29 tcgtcgtttt gacgttttgt cgtt 24 <210> SEQ ID NO 30
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(2)
<223> OTHER INFORMATION: where the internucleotide linkage is
a phosphorothioate internucleotide linkage <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (3)..(10)
<223> OTHER INFORMATION: where the internucleotide linkage is
a phosphorothioate internucleotide linkage <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (11)..(18)
<223> OTHER INFORMATION: where the internucleotide linkage is
a phosphorothioate internucleotide linkage <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (19)..(21)
<223> OTHER INFORMATION: where the internucleotide linkage is
a phosphorothioate internucleotide linkage <400> SEQUENCE: 30
tcgttttgac gttttgtcgt t 21 <210> SEQ ID NO 31 <211>
LENGTH: 13 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(2) <223>
OTHER INFORMATION: where the internucleotide linkage is a
phosphorothioate internucleotide linkage <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (3)..(10)
<223> OTHER INFORMATION: where the internucleotide linkages
are phosphorothioate internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (11)..(13)
<223> OTHER INFORMATION: where the internucleotide linkages
are phosphorothioate internucleotide linkages <400> SEQUENCE:
31 tcgttttgac gtt 13 <210> SEQ ID NO 32 <211> LENGTH:
24 <212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(2) <223> OTHER
INFORMATION: where the internucleotide linkage is a
phosphorothioate internucleotide linkage <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (3)..(5)
<223> OTHER INFORMATION: where the internucleotide linkages
are phosphorothioate internucleotide linkages <220>
FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (6)..(9)
<223> OTHER INFORMATION: where the internucleotide linkages
are phosphorothioate internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (10)..(13)
<223> OTHER INFORMATION: where the internucleotide linkages
are phosphorothioate internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (14)..(21)
<223> OTHER INFORMATION: where the internucleotide linkages
are phosphorothioate internucleotide linkages <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (22)..(24)
<223> OTHER INFORMATION: where the internucleotide linkages
are phosphorothioate internucleotide linkages <400> SEQUENCE:
32 tcgtcgtttt gacgttttgt cgtt 24 <210> SEQ ID NO 33
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(2)
<223> OTHER INFORMATION: wherein the internucleotide linkage
is a phosphorothioate linkage <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (3)..(5) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (6)..(9) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (10)..(13) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(17) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (18)..(21) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (22)..(24) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <400> SEQUENCE: 33 tcgtcgtttt
gacgttttgt cgtt 24 <210> SEQ ID NO 34 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(2) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a
phosphorothioate linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(5) <223> OTHER
INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (6)..(7) <223>
OTHER INFORMATION: wherein the internucleotide linkage is a
phosphorothioate linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (8)..(9) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a
phosphorothioate linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (10)..(11) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a
phosphorothioate linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (12)..(13) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a
phosphorothioate linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (14)..(15) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a
phosphorothioate linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (16)..(17) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a
phosphorothioate linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (18)..(19) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a
phosphorothioate linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (20)..(21) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a
phosphorothioate linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (22)..(24) <223> OTHER
INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <400> SEQUENCE: 34 tcgtcgtttt
gacgttttgt cgtt 24 <210> SEQ ID NO 35 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 35 tcgtcgtttt gacgttttgt cgtt
24 <210> SEQ ID NO 36 <211> LENGTH: 22 <212>
TYPE: DNA <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(3) <223> OTHER INFORMATION: wherein the
internucleotide linkages are phosphorothioate linkages <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(4)..(11) <223> OTHER INFORMATION: wherein the
internucleotide linkages are phosphorothioate linkages <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(12)..(19) <223> OTHER INFORMATION: wherein the
internucleotide linkages are phosphorothioate linkages <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(20)..(22) <223> OTHER INFORMATION: wherein the
internucleotide linkages are phosphorothioate linkages <400>
SEQUENCE: 36 gtcgttttga cgttttgtcg tt 22 <210> SEQ ID NO 37
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(2)
<223> OTHER INFORMATION: wherein the internucleotide linkage
is a phosphorothioate linkage <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (3)..(5) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (6)..(13) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(21) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <400> SEQUENCE: 37 tcgtcgtttt
gacgttttgt c 21 <210> SEQ ID NO 38 <211> LENGTH: 13
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(2) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a
phosphorothioate linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(5) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a
phosphorothioate linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (6)..(13) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a
phosphorothioate linkage <400> SEQUENCE: 38 tcgtcgtttt gac 13
<210> SEQ ID NO 39 <211> LENGTH: 16 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(8)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (9)..(16) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <400> SEQUENCE: 39 gttttgacgt
tttgtc 16 <210> SEQ ID NO 40 <211> LENGTH: 19
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(8) <223> OTHER
INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (9)..(16) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (17)..(19) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <400> SEQUENCE: 40 gttttgacgt
tttgtcgtt 19 <210> SEQ ID NO 41 <211> LENGTH: 14
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(3) <223> OTHER
INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(11) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (12)..(14) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <400> SEQUENCE: 41 gtcgttttga cgtt
14 <210> SEQ ID NO 42 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(2) <223> OTHER INFORMATION: wherein the
internucleotide linkage is a phosphorothioate linkage <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(3)..(10) <223> OTHER INFORMATION: wherein the
internucleotide linkages are phosphorothioate linkages <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(11)..(18) <223> OTHER INFORMATION: wherein the
internucleotide linkages are phosphorothioate linkages <400>
SEQUENCE: 42 tcgttttgac gttttgtc 18 <210> SEQ ID NO 43
<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(3)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(11) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (12)..(19) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <400> SEQUENCE: 43 gtcgttttga
cgttttgtc 19 <210> SEQ ID NO 44 <211> LENGTH: 11
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(3) <223> OTHER
INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(11) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <400> SEQUENCE: 44 gtcgttttga c 11
<210> SEQ ID NO 45 <211> LENGTH: 23 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (2)..(4) <223> OTHER INFORMATION: wherein the
internucleotide linkages are phosphorothioate linkages <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(5)..(12) <223> OTHER INFORMATION: wherein the
internucleotide linkages are phosphorothioate linkages <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(13)..(20) <223> OTHER INFORMATION: wherein the
internucleotide linkages are phosphorothioate linkages <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(21)..(23) <223> OTHER INFORMATION: wherein the
internucleotide linkages are phosphorothioate linkages <400>
SEQUENCE: 45 cgtcgttttg acgttttgtc gtt 23 <210> SEQ ID NO 46
<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(11)
<223> OTHER INFORMATION: wherein the internucleotide linkages
are phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (14)..(23) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <400> SEQUENCE: 46 tcgatcgttt
ttcgtgcgtt ttt 23 <210> SEQ ID NO 47 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(7) <223> OTHER
INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (8)..(15) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (16)..(20) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <400> SEQUENCE: 47 tcgcgacgtt
cgcgcgcgcg 20 <210> SEQ ID NO 48 <211> LENGTH: 19
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(2) <223> OTHER
INFORMATION: wherein the internucleotide linkage is a
phosphorothioate linkage <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(6) <223> OTHER
INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(10) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (11)..(19) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages
<400> SEQUENCE: 48 tcggacgttc ggcgcgccg 19 <210> SEQ ID
NO 49 <211> LENGTH: 12 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
49 cgacgttcgt cg 12 <210> SEQ ID NO 50 <211> LENGTH: 13
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 50 cggcgccgtg ccg 13
<210> SEQ ID NO 51 <211> LENGTH: 12 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 51 ccccccgggg gg 12 <210> SEQ ID NO 52
<211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 52
ggggggcccc cc 12 <210> SEQ ID NO 53 <211> LENGTH: 10
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 53 cccccggggg 10 <210>
SEQ ID NO 54 <211> LENGTH: 10 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 54 gggggccccc 10 <210> SEQ ID NO 55
<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(2)
<223> OTHER INFORMATION: wherein the internucleotide linkage
is a phosphorothioate linkage <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (3)..(5) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (6)..(8) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (9)..(12) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (13)..(17) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (18)..(23) <223>
OTHER INFORMATION: wherein the internucleotide linkages are
phosphorothioate linkages <400> SEQUENCE: 55 tgctcgtcgt
tcggcgcgcg ccg 23 <210> SEQ ID NO 56 <211> LENGTH: 34
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(28) <223> OTHER
INFORMATION: where n is any nucleotide and where any one or more n
may be absent <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (29)..(30) <223> OTHER
INFORMATION: where n is any nucleotide <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (33)..(34)
<223> OTHER INFORMATION: where n is any nucleotide
<400> SEQUENCE: 56 tcnnnnnnnn nnnnnnnnnn nnnnnnntnn cgnn
34
* * * * *